Method for regulating immune function in primates using the Foxp3 protein

ABSTRACT

Isolated nucleic acid molecules are provided which encode Fkh sf , as well as mutant forms thereof. Also provided are expression vectors suitable for expressing such nucleic acid molecules, and host cells containing such expression vectors. Also provided are pharmaceutical compounds and methods of identifying such compounds that can modulate the immune system. In addition are provided methods for identifying proteins regulated by Scurfin and proteins that induce or inhibit Foxp3 expression.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 60/333,409 filed Nov. 26, 2001 and 60/289,654 filed May8, 2001, where these applications are incorporated herein by referencein their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to pharmaceutical products andmethods and, more specifically, to methods for identifying compoundswhich can modulate the immune system, further, to methods foridentifying proteins regulated by Scurfin and those that induce orinhibit Foxp3 expression.

2. Description of the Related Art

A number of autoimmune diseases, such as Inflammatory Bowel Disease,Multiple Sclerosis, rheumatoid Arthritis, and Asthma, involve immunedysregulation. In all these diseases, subsets of T cells arehyper-activated and contribute to an immune reaction towards self. Inrecent years, mice with mutations in CD95, CD95-ligand, CTLA-4 or TGF-βhave proven useful for dissecting a number of pathways involved in Tcell regulation and immune system homeostasis. Mice with mutations inany one if the above genes have profoundly altered immune responses,attributed to a failure to control T cell function.

T cell activation in the periphery involves signaling via the T cellreceptor and CD28 costimulation (reviewed in Bluestone, J. A., Immunity2:555-559 (1995); Jenkins, M. K., Immunity 1:443-448 (1994); Rudd, C.E., Immunity 4:527-534 (1996)). Down regulation of peripheral T cellresponses involves several pathways. Some of these include apoptosismediated by members of the TNFR family, including CD95 and its ligand,activation induced death due to cytokine withdrawal, and negativesignaling through CTLA-4 (CD152) (Lenardo et al., Ann. Rev. Immun.17:221-253 (1999); Oosterwegel et al., Curr. Opin. Immun. 11:294-300(1999); Saito, T., Curr. Opin. Immun. 10:313-321 (1998); Wallach et al.,Ann. Rev. Immun. 17:331-367 (1999)). Mutations or expression of dominantnegative forms of some of these genes have proven their critical role inthe regulation of peripheral T cell responses. Mutations in CD95, CD95L,TGF-β or CTLA-4 lead to progressive autoimmune lymphoproliferativedisorders (Kulkarni et al., Proc. Nat'l. Acad. Sci. USA 90:770-774(1993); Shull et al., Nature 359:693-699 (1992); Takahashi et al., Cell76:969-976 (1994); Tivol et al., Immunity 3:541-547 (1995);Watanbe-Fukunaga et al., Nature 356:314-347 (1992); Waterhouse et al.,Science 270:985-988 (1995)). More recent data suggests that regulationof T cell activity by CD4⁺CD25⁺ regulatory T cells is also important formaintaining peripheral T cell tolerance (Roncarolo et al., Curr. Opin.Immun. 12:676-683 (2000); Sakaguchi, S., Cell 101:455-458 (2000);Shevach, E. M., Ann. Rev. Immun. 18:423-449 (2000)). Depletion of suchregulatory T cells from normal animals leads to development of variousautoimmune diseases and the adoptive transfer of these regulatory cellscan also prevent disease in vivo in a number of systems (Asano et al.,J. Exp. Med. 184:387-396 (1996); Sakaguchi et al., J. Immun.155:1151-1164 (1995); Suri-Payer et al., J. Immun. 160:1212-1218(1998)).

The specific mechanism by which regulatory T cells (T-reg cells) mediatetheir suppressive effect is currently unclear. While TGFB and IL-10 canmediate suppressive effects, and blocking these cytokines eliminatessuppression in some in vivo models, there is good evidence to indicateother molecules are also involved. Mounting evidence indicates a rolefor CD152 in the activation and/or function of CD4⁺CD25⁺ T cells (Readet al., J. Exp. Med. 192:295-302 (2000); Takahashi et al., J. Exp. Med.192:303-310 (2000)). Intriguingly, several studies suggest thatsignaling through CD152 results in the induction of TGFB (Chen et al.,J. Exp. Med. 188:1849-1857 (1998); Gomes et al., J. Immunol.164:2001-2008 (2000); Kitani et al., J. Immunol. 165:691-702 (2000)),providing a potential link between TGFB-mediated inhibition and theinhibitory activity of CD4⁺CD25⁺ cells.

The X-linked lymphoproliferative disease observed in the scurfy (sf)mouse, a spontaneous mutant animal that shares many characteristics withthe pathogenesis seen in targeted deletions of CTLA-4 (Tivol et al.,Immunity 3:541-547 (1995); Waterhouse et al., Science 270:985-988(1995)) as well as TGF-β (Kulkarni et al., Proc. Nat'l. Acad. Sci. USA90:770-774 (1993); Shull et al., Nature 359:693-699 (1992)), includingdeath by three weeks of age (Godfrey et al., Am. J. Pathol. 145:281-286(1994); Godfrey et al., Proc. Nat'l. Acad. Sci. USA 88:5528-5532 (1991);Godfrey et al., Am. J. Pathol. 138:1379-1387 (1991); Kanangat et al.,Eur. J. Immunol. 26:161-165 (1996); Lyon et al., Proc. Nat'l. Acad. Sci.USA 87:2433-2437 (1990)). In sf animals, disease is mediated by CD4⁺ Tcells, and these cells exhibit an activated phenotype both in vivo andin vitro (Blair et al., J. Immunol. 153:3764-774 (1994)). The specificmutation responsible for the disease has been recently cloned and thegene shown to be a new member of the forkhead family of transcriptionfactors (Brunkow et al., Nature Genetics 27:68-72 (2001)). The gene hasbeen designated Foxp3 and the protein product, scurfin. Mutations in theorthologous human gene cause a similar lymphoproliferative disorderamong affected male progeny, which if left untreated is generally fatal(Bennett et al., Nature Genetics 27:20-21 (2001); Chatila et al., JM2,J. Clin. Invest 106:R75-81 (2000); Wildin et al., Nature Genetics27:18-20 (2001)).

The present invention discloses methods and compositions useful fordiagnosing scurfy-related diseases, more specifically, to methods foridentifying compounds which can modulate the immune system, further, tomethods for identifying proteins regulated by Scurfin and those thatinduce or inhibit Foxp3 expression

BRIEF SUMMARY OF THE INVENTION

The present invention relates generally to the discovery of novel geneswhich, when mutated, results in a profound lymphoproliferative disorder.In particular, a mutant mouse, designated ‘Scurfy’, was used to identifythe gene responsible for this disorder through backcross analysis,physical mapping and large-scale DNA sequencing. Analysis of thesequence of this gene indicated that it belongs to a family of relatedgenes, all containing a winged-helix DNA binding domain.

Thus, within one aspect of the invention isolated nucleic acid moleculesare provided which encode FKH^(sf) or Fkh^(sf), including mutant formsthereof. Within certain embodiments, Fkh^(sf) of any type may be from awarm-blooded animal, such as a mouse or human. Within furtherembodiments, isolated nucleic acid molecules are provided wherein thenucleic acid molecule is selected from the group consisting of (a) anucleic acid molecule that encodes an amino acid sequence comprising SEQID NOs:2 or 4, (b) a nucleic acid molecule that hybridizes understringent conditions to a nucleic acid molecule having the nucleotidesequence of SEQ ID NOs:1 or 3, or its complement, and (c) a nucleic acidmolecule that encodes a functional fragment of the polypeptide encodedby either (a) or (b). Preferably, the nucleic acid molecule is not JM2.Within related aspects, vectors (including expression vectors), andrecombinant host cells are also provided, as well as proteins which areencoded by the above-noted nucleic acid molecules. Further, fusionproteins are also provided which combine at least a portion of theabove-described nucleic acid molecules with the coding region of anotherprotein. Also provided are oligonucleotide fragments (including probesand primers) which are based upon the above sequence. Such fragments areat least 8, 10, 12, 15, 20, or 25 nucleotides in length, and may extendup to 100, 200, 500, 1000, 1500, or, 2000 nucleotides in length.

Within other aspects methods of using the above noted expression vectorfor producing a Fkh^(sf) protein (of any type) are provided, comprisingthe general steps of (a) culturing recombinant host cells that comprisethe expression vector and that produce Fkh^(sf) protein, and (b)isolating protein from the cultured recombinant host cells.

Also provided are antibodies and antibody fragments that specificallybind to Fkh^(sf) proteins. Representative examples of such antibodiesinclude both polyclonal and monoclonal antibodies (whether obtained froma murine hybridoma, or derived into human form). Representative examplesof antibody fragments include F(ab′)₂, F(ab)₂, Fab′, Fab, Fv, sFv, andminimal recognition units or complementarity determining regions.

Within yet other aspects, methods are provided for detecting thepresence of a Fkh^(sf) nucleic acid sequence in a biological sample froma subject, comprising the steps of (a) contacting a Fkh^(sf) specificnucleic acid probe under hybridizing conditions with either (i) testnucleic acid molecules isolated from said biological sample, or (ii)nucleic acid molecules synthesized from RNA molecules, wherein the proberecognizes at least a portion of nucleotide sequence of SEQ ID NOs:1 or3, and (b) detecting the formation of hybrids of the nucleic acid probeand (i) or (ii).

Within another related embodiment, methods are provided for detectingthe presence of an Fkh^(sf), or a mutant form thereof, in a biologicalsample, comprising the steps of: (a) contacting a biological sample withan anti-Fkh^(sf) antibody or an antibody fragment, wherein thecontacting is performed under conditions that allow the binding of theantibody or antibody fragment to the biological sample, and (b)detecting any of the bound antibody or bound antibody fragment.

Within other aspects of the invention, methods are provided forintroducing Fkh^(sf) nucleic acid molecules to an animal, comprising thestep of administering a Fkh^(sf) nucleic acid molecule as describedherein to an animal (e.g., a human, monkey, dog, cat, rat, or, mouse).Within one embodiment, the nucleic acid molecule is contained within andexpressed by a viral vector (e.g., a vector generated at least in partfrom a retrovirus, adenovirus, adeno-associated virus, herpes virus, or,alphavirus). Within another embodiment the nucleic acid molecule isexpressed by, or contained within a plasmid vector. Such vectors may beadministered either in vivo, or ex vivo (e.g., to hematopoietic cellssuch as T cells).

Within other embodiments, transgenic non-human animals are providedwherein the cells of the animal express a transgene that contains asequence encoding Fkh^(sf) protein.

In one preferred embodiment, a method is provided for regulating animmune function in a primate. The method comprises inserting a pluralityof nucleic acid sequences that encode the Foxp3 protein into thelymphocytes of the primate; placing the nucleic acid sequence under thecontrol of cytokine c; and activating expression of the nucleic acidsequences to increase the amount of the Foxp3 protein in the primatewith cytokine c.

Accordingly, it is an object of the present invention to provide anassay for use in identifying agents that alter expression of Foxp3.Specifically, an assay is provided to measure the induction orinhibition of Foxp3 under varying conditions. The expression alteringagents include small molecules, peptides, polynucleotides, cytokines,antibodies and Fab′ fragments.

In one preferred embodiment, a method is provided for identifying acompound that modulates the level of expression of scurfin. The methodcomprises providing a composition comprising a reporter gene ligated toa scurfin promoter; contacting the composition with a test compound;determining the level of reporter gene expression; and comparing thelevel of reporter gene expression in (c) with the predetermined level ofexpression and thereby determining if the test compound modulates theexpression of scurfin.

In a preferred embodiment, the compound decreases the level of scurfinexpression.

In another embodiment, the compound increases the level of scurfin.

In one embodiment the test compound is selected from the groupconsisting of: a monoclonal antibody, a polyclonal antibody, a peptide,and a small molecule.

In another embodiment the test compound is selected from the groupconsisting of an organic molecule, a natural product, a peptide, anoligosaccharide, a nucleic acid, a lipid, an antibody or bindingfragment thereof, and a cell.

In yet another embodiment, the test compound is from a library ofcompounds.

With other embodiments, the library is selected from the groupconsisting of a random peptide library, a natural products library, acombinatorial library, an oligosaccharide library and a phage displaylibrary.

In one preferred embodiment, a method is provided for suppressing animmune response comprising contacting T cells of the mammal with acompound that increases scurfin expression in the T cell, wherein animmune response is suppressed.

In one preferred embodiment, a method is provided for enhancing animmune response comprising contacting T cells with a compound thatdecreases scurfin expression in the T cell, wherein an immune responseis enhanced.

Within another related embodiment, a method for inhibiting an autoimmuneresponse in a subject, wherein the method comprises administering to thesubject a compound which increases scurfin expression, therebyinhibiting an autoimmune response by the subject.

In a related embodiment the autoimmune response is selected from thegroup consisting of Inflammatory Bowel Disease, Psoriasis, Diabetes,Multiple Sclerosis, Rheumatoid Arthritis, and Asthma.

In one preferred embodiment, a method is provided for enhancing animmune response to a disease in a subject, wherein the method comprisesadministering to the subject a compound which decreases scurfinexpression, thereby treating the disease in the subject.

In a related embodiment, a method is provided for enhancing an immuneresponse to HIV or cancer in a subject, wherein the method comprisesadministering to the subject a compound which decreases scurfinexpression, thereby treating HIV and cancer.

In one preferred embodiment, a method for inhibiting graft versus hostdisease in a subject wherein the method comprises administering to thesubject a compound that increases scurfin expression, thereby inhibitingtissue transplant rejection by the subject.

In one preferred embodiment, a method is provided for inhibiting anautoimmune response in a patient comprising. The method comprising:isolating T cells from the patient; transducing the T cells with thescurfin gene; expanding the transduced T cells; and reintroducing thetransduced T cells into said patient, wherein an autoimmune disease inthe patient is inhibited.

In a related embodiment, a method is provided for inhibiting anautoimmune response in a patient comprising. The method comprising:isolating CD4⁺CD25+ regulatory T cells from the patient; transducing theCD4⁺CD25+ regulatory T cells with the scurfin gene; expanding thetransduced CD4⁺CD25+ regulatory T cells; and reintroducing thetransduced CD4⁺CD25+ regulatory T cells into the patient, wherein anautoimmune disease in the patient is inhibited.

In one preferred embodiment, a method is provided for inhibiting anautoimmune response in a patient, wherein the autoimmune disease isselected from the group consisting of Inflammatory Bowel Disease,Multiple Sclerosis, Rheumatoid Arthritis, Psoriasis, Diabetes andAsthma. The method comprising: isolating T cells from the patient;transducing the T cells with the scurfin gene; expanding the transducedT cells; and reintroducing the transduced T cells into said patient,wherein an autoimmune disease in the patient is inhibited.

In one preferred embodiment, a method is provided for inhibiting anautoimmune response in a patient comprising. The method comprising:isolating T cells from the patient; transducing the T cells with thescurfin gene contained in a retroviral vector; expanding the transducedT cells; and reintroducing the transduced T cells into said patient,wherein an autoimmune disease in the patient is inhibited.

In yet another preferred embodiment, a method for enhancing an immuneresponse to a disease in a patient is provided. The method comprising:isolating T cells from the patient; transfecting the T cells with a testcompound that inhibits scurfin expression; expanding the transfected Tcells; and reintroducing the transfected T cells into said patient,wherein an immune response to a disease in the patient is enhanced.

In yet another preferred embodiment, a method for enhancing an immuneresponse to a HIV and cancer in a patient is provided. The methodcomprising: isolating T cells from the patient; transfecting the T cellswith a test compound that inhibits scurfin expression; expanding thetransfected T cells; and reintroducing the transfected T cells into saidpatient, wherein an immune response to HIV or cancer in the patient isenhanced.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, various references are set forth herein whichdescribe in more detail certain procedures or compositions (e.g.,plasmids, etc.), and are therefore incorporated by reference in theirentirety.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIGS. 1A and 1B depict a nucleotide sequence of mouse Fkh^(sf) cDNA (SEQID NO:1); translation is predicted to initiate at position 259 andterminate at position 1546.

FIG. 2 depicts the amino acid sequence of mouse Fkh^(sf) (SEQ ID NO:2).

FIGS. 3A and 3B depict a nucleotide sequence of 1735 bp corresponding tohuman FKH^(sf) cDNA (SEQ ID NO: 3; including a 1293 bp coding region);translation is predicted to initiate at position 55 and terminate atposition 1348.

FIG. 4 depicts the sequence of a 431 amino acid human FKH^(sf) protein(SEQ ID NO: 4).

FIG. 5 diagrammatically depicts a vector for generation of FKH^(sf)transgenic mice.

FIG. 6 is a photograph which demonstrates that the FKH^(sf) transgenecorrects the defect in scurfy animals.

FIG. 7 is a diagram which shows that FKH^(sf) tg mice have reduced lymphnode cells, as compared to normal cells.

FIG. 8 is a diagram which shows that FKH^(sf) transgenic mice respondpoorly to in vitro stimulation.

FIG. 9 is a comparison of FKH^(sf) and JM2 cDNAs.

FIG. 10 compares homology in various regions of human FKH^(sf) andmurine Fkh^(sf).

FIG. 11A is a graph monitoring the weight of both scurfy and wild-typemice. The mice were monitored for weight loss at regular intervals for10 weeks. Each data point is an average of 3 mice except after week 5when one of the three mice died in sf CD4 transfer group (indicated byan arrow on the graph). The data is representative of more than 3independent experiments.

FIG. 11B is a photograph of a tissue section. Large intestines fromC3H/SCID mice receiving either sf T cells (left panel) or a mixture ofWT and sf T cells (right panel) were fixed in formalin, sectioned andprocessed for hematoxylin and eosin staining.

FIG. 11C is a graph depicting IL-4 production from 5×10⁴ PBMC fromC3H/SCID mice receiving either sf CD4⁺ T cells or a mixture of WT and sfCD4⁺ T cells or WT CD4⁺ T cells were stimulated with 5 μg/ml anti-CD3and 1 μg/ml anti-CD28 immobilized onto round bottom plates. Supernatantswere harvested at 48 h and IL-4 levels were measured by ELISA.

FIGS. 12A and 12B are graphs depicting the weight loss of mice treatedwith either CD4+CD25+ or CD4⁺CD25− T-regulatory cells. CD4+CD25+T-regulatory subset mediates the suppression of disease caused by sf Tcells in vivo. A mixture of 4×10⁶ sf T cells and varying numbers ofwildtype CD4+CD25+ (a) or CD4+CD25− (b) T cells was transferred intoC3/SCID mice via tail-vein injection. These mice were monitored forweight loss over a period of time. Each data point is an average of 3mice except sf CD4 transfer group and sf CD4+1.1×10⁶ CD4+CD25− T cellswhich have 2 mice each in the group. Also, arrows on the graph indicatemice that died or were sacrificed due to disease progression.

FIG. 13 depicts a graph of a proliferation assay that determines thesuppressor activity of CD4+CD25+ T regulatory cells. Sf CD4+ T cells canbe inhibited by CD4+CD25+ T-regulatory cells in vitro. 5×10⁴ WT or sfCD4+ T cells were stimulated with anti-CD3 (1 μg/ml) and 5×10⁴ mitomycinC treated Thy-1⁻ APC. CD4⁺CD25⁺ T-regulatory cells were added at variousratios to the assay. The cells were cultured for 72 h and pulsed with[³H] thymidine for final 8 hrs of the culture. Data is mean oftriplicates.

FIG. 14 A depicts a graph of a proliferation assay in which 5×10⁴ WT orsf CD4+ T cells were stimulated with immobilized anti-CD3. TGF-β wasadded at a final concentration of 2.5 ng/ml at the beginning of theassay. The cells were cultured for 72 h and pulsed with [³H] thymidinefor final 8 hrs of the culture. Data is mean of triplicates.

FIG. 14B depicts a graph of a proliferation assay in which 5×10⁴ WT orsf CD4+ T cells were stimulated with immobilized anti-CD3 (varyingconcentrations) and anti-CD28 (1 μg/ml). TGF-β was added at a finalconcentration of 2.5 ng/ml at the beginning of the assay. The cells werecultured for 72 h and pulsed with [³H] thymidine for final 8 hrs of theculture. Data is mean of triplicates.

FIGS. 15A and 15B are graphs examining Foxp3 expression in cDNA samplesfrom various cell subsets using a real-time RT-PCR method in which Dad1served as an endogenous reference gene. Normalized Foxp3 values werederived from the ratio of Foxp3 expression to Dad1 expression.

FIG. 16 depicts the level of CD25 surface expression on CD4+ T cellsfrom WT animals, Foxp3 transgenic animals, and scurfy animals. Lymphnode cells from sf, Foxp3 transgenic or littermate controls wereexamined for the expression of CD25 expression on CD4⁺ T cells. Data isrepresentative of six individual mice examined.

FIG. 17 is a graph depicting the level of proliferation in 5×10⁴ WT CD4+T cells were stimulated with anti-CD3 (1 μg/ml) and 5×10⁴ mitomycin Ctreated Thy-1⁻ APC. CD4⁺CD25⁺ T-regulatory cells from WT or Foxp3transgenics were added at various ratios to the assay. The cells werecultured for 72 h and pulsed with [³H] thymidine for final 8 hrs of theculture. Data is mean or triplicates.

FIG. 18 is a FACS plot evaluating the expression of surface markersassociated with T regulatory cells and the suppressive activity of thesecells.

FIG. 19 is a graph depicting the level of T cell inhibition in freshlyisolated CD4⁺CD25⁻ T cells from Foxp3 transgenic as tested in T-regassays.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to the discovery that the scurfin protein isinvolved in the generation and/or activity of the CD4⁺CD25⁺ subset ofregulatory T cells. Foxp3 expression is directly correlated with cellsof this regulatory phenotype and its expression is uniquely increasedupon activation of this specific subset. Mutant (sf) animals appear tolack this subset, whereas Foxp3 transgenic animals appear to possess anincreased percentage of CD4⁺CD25⁺ cells. Further, while the CD4⁺CD25⁺subset from transgenic animals does not appear inhibitory on a per cellbasis, the expression of Foxp3 is still elevated in this subset relativeto their CD25− counterparts. Interestingly, overexpression of Foxp3 inCD4⁺CD25⁻ T cells confers suppressive activity on these cells, althoughthey remain less effective than CD4⁺CD25⁺ T cells. Overall, the datasuggest that the recently described transcription factor, scurfin, is acritical regulator of immune cell function and may work primarilythrough the generation and/or activity of CD4⁺CD25⁺ regulatory T cells.

The results from the Examples indicate that expression of scurfin (Foxp3gene) can downregulate the immune system in part through regulatory T(T-reg) cell activity. Consequentially, if the expression of theendogenous Foxp3 gene can be induced in T cells it can be used todownregulate the immune response in a variety of autoimmune diseasessuch as Inflammatory Bowel Disease, Multiple Sclerosis, RheumatoidArthritis, Psoriasis, Diabetes, and Asthma or in other scenarios such asGraft versus Host disease. Furthermore, scurfin expression can bedown-regulated to activate the immune system in cancer or AIDS.

DEFINITIONS

Prior to setting forth the Invention in detail, it may be helpful to anunderstanding thereof to set forth definitions of certain terms and tolist and to define the abbreviations that will be used hereinafter.

“Scurfy” refers to an inherited disease in mice which exhibit a severelymphoproliferative disorder (see, e.g., Lyon et al., Proc. Natl. Acad.Sci. USA 87:2433, 1990). The responsible gene (mutant forms of which areresponsible for the disease) is shown in Sequence I.D. Nos. 1 and 3.

“Foxp3” refers to the forkhead domain-containing gene, which is mutatedin the scurfy mouse mutant. “Foxp3” refers to the protein encoded by themouse Foxp3 gene. “FOXP3” refers to the human ortholog of the murineFoxp3 gene. “FOXP3” refers to the protein encoded by the human FOXP3gene. The cDNA sequences for murine Foxp3 and human FOXP3 are disclosedin U.S. patent application Ser. No. 09/372,668 wherein the mouse scurfygene is designated Fkh^(sf) and the human ortholog is designatedFKH^(sf). The genomic sequence for human FOXP3 is disclosed in GenbankAccession No. AF235087. Genbank Accession No. AF235097 and U.S. patentapplication Ser. No. 09/372,668 are incorporated by reference in theirentireties for all purposes.

“Molecule” should be understood to include proteins or peptides (e.g.,antibodies, recombinant binding partners, peptides with a desiredbinding affinity), nucleic acids (e.g., DNA, RNA, chimeric nucleic acidmolecules, and nucleic acid analogues such as PNA), and organic orinorganic compounds.

“Nucleic acid” or “nucleic acid molecule” refers to any ofdeoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides,fragments generated by the polymerase chain reaction (PCR), andfragments generated by any of ligation, scission, endonuclease action,and exonuclease action. Nucleic acids can be composed of monomers thatare naturally-occurring nucleotides (such as deoxyribonucleotides andribonucleotides), or analogs of naturally-occurring nucleotides (e.g.,α-enantiomeric forms of naturally-occurring nucleotides), or acombination of both. Modified nucleotides can have modifications insugar moieties and/or in pyrimidine or purine base moieties. Sugarmodifications include, for example, replacement of one or more hydroxylgroups with halogens, alkyl groups, amines, and azido groups, or sugarscan be functionalized as ethers or esters. Moreover, the entire sugarmoiety can be replaced with sterically and electronically similarstructures, such as aza-sugars and carbocyclic sugar analogs. Examplesof modifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleicacid” also includes so-called “peptide nucleic acids,” which comprisenaturally-occurring or modified nucleic acid bases attached to apolyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

“Isolated nucleic acid molecule” is a nucleic acid molecule that is notintegrated in the genomic DNA of an organism. For example, a DNAmolecule that corresponds to a gene that has been separated from thegenomic DNA of a eukaryotic cell is an isolated DNA molecule. Anotherexample of an isolated nucleic acid molecule is a chemically-synthesizednucleic acid molecule that is not integrated in the genome of anorganism.

“Promoter” is a nucleotide sequence that directs the transcription of astructural gene. Typically, a promoter is located in the 5′ region of agene, proximal to the transcriptional start site of a structural gene.If a promoter is an inducible promoter, then the rate of transcriptionincreases in response to an inducing agent. In contrast, the rate oftranscription is not regulated by an inducing agent if the promoter is aconstitutive promoter.

“Vector” refers to an assembly which is capable of directing theexpression of desired protein. The vector must include transcriptionalpromoter elements which are operably linked to the genes of interest.The vector may be composed of either deoxyribonucleic acids (“DNA”),ribonucleic acids (“RNA”), or a combination of the two (e.g., a DNA-RNAchimeric). Optionally, the vector may include a polyadenylationsequence, one or more restriction sites, as well as one or moreselectable markers such as neomycin phosphotransferase or hygromycinphosphotransferase. Additionally, depending on the host cell chosen andthe vector employed, other genetic elements such as an origin ofreplication, additional nucleic acid restriction sites, enhancers,sequences conferring inducibility of transcription, and selectablemarkers, may also be incorporated into the vectors described herein.

“Isolated” in the case of proteins or polypeptides, refers to moleculeswhich are present in the substantial absence of other biologicalmacromolecules, and appear nominally as a single band on SDS-PAGE gelwith coomassie blue staining. “Isolated” when referring to organicmolecules means that the compounds are greater than 90% pure utilizingmethods which are well known in the art (e.g., NMR, melting point).

“Cloning vector” refers to nucleic acid molecules, such as a plasmid,cosmid, or bacteriophage, that has the capability of replicatingautonomously in a host cell. Cloning vectors typically contain one or asmall number of restriction endonuclease recognition sites at whichforeign nucleotide sequences can be inserted in a determinable fashionwithout loss of an essential biological function of the vector, as wellas nucleotide sequences encoding a marker gene that is suitable for usein the identification and selection of cells transformed with thecloning vector. Marker genes typically include genes that providetetracycline resistance or ampicillin resistance.

“Expression vector” refers to a nucleic acid molecule encoding a genethat is expressed in a host cell. Typically, gene expression is placedunder the control of a promoter, and optionally, under the control of atleast one regulatory element. Such a gene is said to be “operably linkedto” the promoter. Similarly, a regulatory element and a promoter areoperably linked if the regulatory element modulates the activity of thepromoter.

“Recombinant host” refers to any prokaryotic or eukaryotic cell thatcontains either a cloning vector or expression vector. This term alsoincludes those prokaryotic or eukaryotic cells that have beengenetically engineered to contain the cloned gene(s) in the chromosomeor genome of the host cell.

In eukaryotes, RNA polymerase II catalyzes the transcription of astructural gene to produce mRNA. A nucleic acid molecule can be designedto contain an RNA polymerase II template in which the RNA transcript hasa sequence that is complementary to that of a specific mRNA. The RNAtranscript is termed an “anti-sense RNA” and a nucleic acid moleculethat encodes the anti-sense RNA is termed an “anti-sense gene.”Anti-sense RNA molecules are capable of binding to mRNA molecules,resulting in an inhibition of mRNA translation.

An “anti-sense oligonucleotide specific for Fkh^(sf)” or a “Fkh^(sf)anti-sense oligonucleotide” is an oligonucleotide having a sequence (a)capable of forming a stable triplex with a portion of the gene, or (b)capable of forming a stable duplex with a portion of an mRNA transcript.Similarly, an “anti-sense oligonucleotide specific for “Fkh^(sf)” or a“Fkh^(sf) anti-sense oligonucleotide” is an oligonucleotide having asequence (a) capable of forming a stable triplex with a portion of theFkh^(sf) gene, or (b) capable of forming a stable duplex with a portionof an mRNA transcript of the Fkh^(sf) gene.

A “ribozyme” is a nucleic acid molecule that contains a catalyticcenter. The term includes RNA enzymes, self-splicing RNAs, self-cleavingRNAs, and nucleic acid molecules that perform these catalytic functions.A nucleic acid molecule that encodes a ribozyme is termed a “ribozymegene.”

Abbreviations: YAC, yeast artificial chromosome; PCR, polymerase chainreaction; RT-PCR, PCR process in which RNA is first transcribed into DNAat the first step using reverse transcriptase (RT); cDNA, any DNA madeby copying an RNA sequence into DNA form. As utilized herein “Fkh^(sf)”refers to the gene product of the Fkh^(sf) gene (irrespective of whetherthe gene is obtained from humans, mammals, or any other warm-bloodedanimal). When capitalized “FKH^(sf)” the gene product (and gene) shouldbe understood to be derived from humans.

As noted above, the present invention relates generally topharmaceutical products and methods and, more specifically, to methodsand compositions useful for diagnosing scurfy-related diseases, as wellas methods for identifying compounds which can modulate the immunesystem.

Thus, as discussed in more detail below this discovery has led to thedevelopment of assays which may be utilized to select molecules whichcan act as agonists, or alternatively, antagonists of the immune system.Furthermore, such assays may be utilized to identify other genes andgene products which are likewise active in modulating the immune system.

Scurfy

Briefly, the present invention is based upon the unexpected discoverythat a mutation in the gene which encodes Fkh^(sf) results in rarecondition (scurfy) characterized by a progressive lymphocyticinfiltration of the lymph nodes, spleen, liver and skin resulting ingross morphological symptoms which include splenomegaly, hepatomegaly,greatly enlarged lymph nodes, runting, exfoliative dermatitis, andthickened malformed ears (Godfrey et al., Amer. J. Pathol. 138:1379,1991; Godfrey et al., Proc. Natl. Acad. Sci. USA 88:5528, 1991). Thisnew member of the winged-helix family represents a novel component ofthe immune system.

Methods which were utilized to discover the gene responsible for scurfyare provided below in Example 1. Methods for cloning the generesponsible for murine scurfy, as well as the human ortholog, areprovided below in Examples 2 and 3. Methods for confirmation of geneidentity and correlation with gene function, as determined usingtransgenic mice, are also provided in the Examples.

Also provided by the present invention are methods for determining thepresence of Fkh^(sf) genes and gene products. Within one embodiment,such methods comprise the general steps of (a) contacting a Fkh^(sf)specific nucleic acid probe under hybridizing conditions with either (i)test nucleic acid molecules isolated from the biological sample, or (ii)nucleic acid molecules synthesized from RNA molecules, wherein the proberecognizes at least a portion of an Fkh^(sf) nucleotide sequence, and(b) detecting the formation of hybrids of said nucleic acid probe and(i) or (ii). A variety of methods may be utilized in order to amplify aselected sequence, including, for example, RNA amplification (seeLizardi et al., Bio/Technology 6:1197-1202, 1988; Kramer et al., Nature339:401-02, 1989; Lomeli et al., Clinical Chem. 35(9):1826-31, 1989;U.S. Pat. No. 4,786,600), and nucleic acid amplification utilizingPolymerase Chain Reaction (“PCR”) (see U.S. Pat. Nos. 4,683,195,4,683,202, and 4,800,159), reverse-transcriptase-PCR and CPT (see U.S.Pat. Nos. 4,876,187, and 5,011,769).

Alternatively, antibodies may be utilized to detect the presence ofFkh^(sf) gene products. More specifically, within one embodiment methodsare provided for detecting the presence of an Fkh^(sf) peptide, or amutant form thereof, in a biological sample, comprising the steps of (a)contacting a biological sample with an anti-Fkh^(sf) antibody or anantibody fragment, wherein said contacting is performed under conditionsthat allow the binding of said antibody or antibody fragment to thebiological sample, and (b) detecting any of the bound antibody or boundantibody fragment.

Such methods may be accomplished in a wide variety of assay formatsincluding, for example, Countercurrent Immuno-Electrophoresis (CIEP),Radioimmunoassays, Radioimmunoprecipitations, Enzyme-LinkedImmuno-Sorbent Assays (ELISA), Dot Blot assays, Inhibition orCompetition assays, and sandwich assays (see U.S. Pat. Nos. 4,376,110and 4,486,530; see also Antibodies: A Laboratory Manual, supra).

Nucleic Acid Molecules, Proteins, and Methods of Producing Proteins

Although various FKH^(sf) or Fkh^(sf) proteins and nucleic acidmolecules (or portions thereof) have been provided herein, it should beunderstood that within the context of the present invention, referenceto one or more of these proteins should be understood to includeproteins of a substantially similar activity. As used herein, proteinsare deemed to be “substantially similar” if: (a) they are encoded by anucleotide sequence which is derived from the coding region of a genewhich encodes the protein (including, for example, portions of thesequence or allelic variations of the sequence); (b) the nucleotidesequence is capable of hybridization to nucleotide sequences of thepresent invention under moderate, high or very high stringency (seeSambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., ColdSpring Harbor Laboratory Press, N.Y., 1989), or has at least 50%, 60%,70%; 75%, 80%, 90%, 95%, or greater homology to the sequences disclosedherein, or, (c) the DNA sequences are degenerate as a result of thegenetic code to the DNA sequences defined in (a) or (b). Further, thenucleic acid molecule disclosed herein includes both complementary andnon-complementary sequences, provided the sequences otherwise meet thecriteria set forth herein. Within the context of the present invention,high stringency means standard hybridization conditions (e.g., 5×SSPE,0.5% SDS at 65° C., or the equivalent). For purpose of hybridization,nucleic acid molecules which encode the amino-terminal domain, zincfinger domain, middle domain, or forkhead domain (see Example 10) may beutilized.

The structure of the proteins encoded by the nucleic acid moleculesdescribed herein may be predicted from the primary translation productsusing the hydrophobicity plot function of, for example, P/C Gene orIntelligenetics Suite (Intelligenetics, Mountain View, Calif.), oraccording to the methods described by Kyte and Doolittle (J. Mol. Biol.157:105-32, 1982).

Proteins of the present invention may be prepared in the form of acidicor basic salts, or in neutral form. In addition, individual amino acidresidues may be modified by oxidation or reduction. Furthermore, varioussubstitutions, deletions, or additions may be made to the amino acid ornucleic acid sequences, the net effect of which is to retain or furtherenhance or decrease the biological activity of the mutant or wild-typeprotein. Moreover, due to degeneracy in the genetic code, for example,there may be considerable variation in nucleotide sequences encoding thesame amino acid sequence.

Other derivatives of the proteins disclosed herein include conjugates ofthe proteins along with other proteins or polypeptides. This may beaccomplished, for example, by the synthesis of N-terminal or C-terminalfusion proteins which may be added to facilitate purification oridentification of proteins (see U.S. Pat. No. 4,851,341, see also, Hoppet al., Bio/Technology 6:1204, 1988.) Alternatively, fusion proteins(e.g., FKH or Fkh-luciferase or FKH or Fkh-GFP) may be constructed inorder to assist in the identification, expression, and analysis of theprotein.

Proteins of the present invention may be constructed using a widevariety of techniques described herein. Further, mutations may beintroduced at particular loci by synthesizing oligonucleotidescontaining a mutant sequence, flanked by restriction sites enablingligation to fragments of the native sequence. Following ligation, theresulting reconstructed sequence encodes a derivative having the desiredamino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific (or segmentspecific) mutagenesis procedures may be employed to provide an alteredgene having particular codons altered according to the substitution,deletion, or insertion required. Exemplary methods of making thealterations set forth above are disclosed by Walder et al. (Gene 42:133,1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, Jan. 1985,12-19); Smith et al. (Genetic Engineering: Principles and Methods,Plenum Press, 1981); and Sambrook et al. (supra). Deletion or truncationderivatives of proteins (e.g., a soluble extracellular portion) may alsobe constructed by utilizing convenient restriction endonuclease sitesadjacent to the desired deletion. Subsequent to restriction, overhangsmay be filled in, and the DNA religated. Exemplary methods of making thealterations set forth above are disclosed by Sambrook et al. (MolecularCloning: A Laboratory Manual, 2d ed., Cold Spring Harbor LaboratoryPress, 1989).

Mutations which are made in the nucleic acid molecules of the presentinvention preferably preserve the reading frame of the coding sequences.Furthermore, the mutations will preferably not create complementaryregions that could hybridize to produce secondary mRNA structures, suchas loops or hairpins, that would adversely affect translation of themRNA. Although a mutation site may be predetermined, it is not necessarythat the nature of the mutation per se be predetermined. For example, inorder to select for optimum characteristics of mutants at a given site,random mutagenesis may be conducted at the target codon and theexpressed mutants screened for indicative biological activity.Alternatively, mutations may be introduced at particular loci bysynthesizing oligonucleotides containing a mutant sequence, flanked byrestriction sites enabling ligation to fragments of the native sequence.Following ligation, the resulting reconstructed sequence encodes aderivative having the desired amino acid insertion, substitution, ordeletion. Mutations may be introduced for purpose of preserving orincreasing activity of the protein, or, for decreasing or disabling theprotein (e.g., mutant Fkh).

Nucleic acid molecules which encode proteins of the present inventionmay also be constructed utilizing techniques of PCR mutagenesis,chemical mutagenesis (Drinkwater and Klinedinst, PNAS 83:3402-06, 1986),by forced nucleotide misincorporation (e.g., Liao and Wise Gene88:107-11, 1990), or by use of randomly mutagenized oligonucleotides(Horwitz et al., Genome 3:112-17, 1989).

The present invention also provides for the manipulation and expressionof the above described genes by culturing host cells containing a vectorcapable of expressing the above-described genes. Such vectors or vectorconstructs include either synthetic or cDNA-derived nucleic acidmolecules encoding the desired protein, which are operably linked tosuitable transcriptional or translational regulatory elements. Suitableregulatory elements may be derived from a variety of sources, includingbacterial, fungal, viral, mammalian, insect, or plant genes. Selectionof appropriate regulatory elements is dependent on the host cell chosen,and may be readily accomplished by one of ordinary skill in the art.Examples of regulatory elements include: a transcriptional promoter andenhancer or RNA polymerase binding sequence, a transcriptionalterminator, and a ribosomal binding sequence, including a translationinitiation signal.

Nucleic acid molecules that encode any of the proteins described abovemay be readily expressed by a wide variety of prokaryotic and eukaryotichost cells, including bacterial, mammalian, yeast or other fungi, viral,insect, or plant cells. Methods for transforming or transfecting suchcells to express foreign DNA are well known in the art (see, e.g.,Itakura et al., U.S. Pat. No. 4,704,362; Hinnen et al., Proc. Natl.Acad. Sci. USA 75:1929-33, 1978; Murray et al., U.S. Pat. No. 4,801,542;Upshall et al., U.S. Pat. No. 4,935,349; Hagen et al., U.S. Pat. No.4,784,950; Axel et al., U.S. Pat. No. 4,399,216; Goeddel et al., U.S.Pat. No. 4,766,075; and Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989; for plantcells see Czako and Marton, Plant Physiol. 104:1067-71, 1994; andPaszkowski et al., Biotech. 24:387-92, 1992).

Bacterial host cells suitable for carrying out the present inventioninclude E. coli, B. subtilis, Salmonella typhimurium, and variousspecies within the genera Pseudomonas, Streptomyces, and Staphylococcus,as well as many other bacterial species well known to one of ordinaryskill in the art. Representative examples of bacterial host cellsinclude DH5α (Stratagene, LaJolla, Calif.).

Bacterial expression vectors preferably comprise a promoter whichfunctions in the host cell, one or more selectable phenotypic markers,and a bacterial origin of replication. Representative promoters includethe β-lactamase (penicillinase) and lactose promoter system (see Changet al., Nature 275:615, 1978), the T7 RNA polymerase promoter (Studieret al., Meth. Enzymol. 185:60-89, 1990), the lambda promoter (Elvin etal., Gene 87:123-26, 1990), the trp promoter (Nichols and Yanofsky,Meth. in Enzymology 101:155, 1983) and the tac promoter (Russell et al.,Gene 20:231, 1982). Representative selectable markers include variousantibiotic resistance markers such as the kanamycin or ampicillinresistance genes. Many plasmids suitable for transforming host cells arewell known in the art, including among others, pBR322 (see Bolivar etal., Gene 2:95, 1977), the pUC plasmids pUC18, pUC19, pUC118, pUC119(see Messing, Meth. in Enzymology 101:20-77, 1983 and Vieira andMessing, Gene 19:259-68, 1982), and pNH8A, pNH16a, pNH18a, andBluescript M13 (Stratagene, La Jolla, Calif.).

Yeast and fungi host cells suitable for carrying out the presentinvention include, among others, Saccharomyces pombe, Saccharomycescerevisiae, the genera Pichia or Kluyveromyces and various species ofthe genus Aspergillus (McKnight et al., U.S. Pat. No. 4,935,349).Suitable expression vectors for yeast and fungi include, among others,YCp50 (ATCC No. 37419) for yeast, and the amdS cloning vector pV3(Turnbull, Bio/Technology 7:169, 1989), YRp7 (Struhl et al., Proc. Natl.Acad. Sci. USA 76:1035-39, 1978), YEp13 (Broach et al., Gene 8:121-33,1979), pJDB249 and pJDB219 (Beggs, Nature 275:104-08, 1978) andderivatives thereof.

Preferred promoters for use in yeast include promoters from yeastglycolytic genes (Hitzeman et al., J. Biol. Chem. 255:12073-080, 1980;Alber and Kawasaki, J. Mol. Appl. Genet. 1:419-34, 1982) or alcoholdehydrogenase genes (Young et al., Hollaender et al. (eds.), in GeneticEngineering of Microorganisms for Chemicals, Plenum, New York, 1982, p.355; Ammerer, Meth. Enzymol. 101:192-201, 1983). Examples of usefulpromoters for fungi vectors include those derived from Aspergillusnidulans glycolytic genes, such as the adh3 promoter (McKnight et al.,EMBO J. 4:2093-99, 1985). The expression units may also include atranscriptional terminator. An example of a suitable terminator is theadh3 terminator (McKnight et al., ibid., 1985).

As with bacterial vectors, the yeast vectors will generally include aselectable marker, which may be one of any number of genes that exhibita dominant phenotype for which a phenotypic assay exists to enabletransformants to be selected. Preferred selectable markers are thosethat complement host cell auxotrophy, provide antibiotic resistance orenable a cell to utilize specific carbon sources, and include leu2(Broach et al., ibid.), ura3 (Botstein et al., Gene 8:17, 1979), or his3(Struhl et al., ibid.). Another suitable selectable marker is the catgene, which confers chloramphenicol resistance on yeast cells.

Techniques for transforming fungi are well known in the literature, andhave been described, for instance, by Beggs (ibid.), Hinnen et al.(Proc. Natl. Acad. Sci. USA 75:1929-33, 1978), Yelton et al. (Proc.Natl. Acad. Sci. USA 81:1740-47, 1984), and Russell (Nature 301:167-69,1983). The genotype of the host cell may contain a genetic defect thatis complemented by the selectable marker present on the expressionvector. Choice of a particular host and selectable marker is well withinthe level of ordinary skill in the art.

Protocols for the transformation of yeast are also well known to thoseof ordinary skill in the art. For example, transformation may be readilyaccomplished either by preparation of spheroplasts of yeast with DNA(see Hinnen et al., PNAS USA 75:1929, 1978) or by treatment withalkaline salts such as LiCl (see Itoh et al., J. Bacteriology 153:163,1983). Transformation of fungi may also be carried out usingpolyethylene glycol as described by Cullen et al. (Bio/Technology 5:369,1987).

Viral vectors include those which comprise a promoter that directs theexpression of an isolated nucleic acid molecule that encodes a desiredprotein as described above. A wide variety of promoters may be utilizedwithin the context of the present invention, including for example,promoters such as MoMLV LTR, RSV LTR, Friend MuLV LTR, adenoviralpromoter (Ohno et al., Science 265:781-84, 1994), neomycinphosphotransferase promoter/enhancer, late parvovirus promoter (Koeringet al., Hum. Gene Therap. 5:457-63, 1994), Herpes TK promoter, SV40promoter, metallothionein Ila gene enhancer/promoter, cytomegalovirusimmediate early promoter, and the cytomegalovirus immediate latepromoter. Within particularly preferred embodiments of the invention,the promoter is a tissue-specific promoter (see e.g., WO 91/02805; EP0,415,731; and WO 90/07936). Representative examples of suitable tissuespecific promoters include neural specific enolase promoter, plateletderived growth factor beta promoter, human alpha1-chimaerin promoter,synapsin I promoter and synapsin II promoter. In addition to theabove-noted promoters, other viral-specific promoters (e.g., retroviralpromoters (including those noted above, as well as others such as HIVpromoters), hepatitis, herpes (e.g., EBV), and bacterial, fungal orparasitic (e.g., malarial)-specific promoters may be utilized in orderto target a specific cell or tissue which is infected with a virus,bacteria, fungus or parasite.

Mammalian cells suitable for carrying out the present invention include,among others: PC12 (ATCC No. CRL1721), N1E-115 neuroblastoma,SK-N-BE(2)C neuroblastoma, SHSY5 adrenergic neuroblastoma, NS20Y andNG108-15 murine cholinergic cell lines, or rat F2 dorsal root ganglionline, COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g., ATCC No. CRL6281; BHK 570 cell line (deposited with the American Type CultureCollection under accession number CRL 10314)), CHO (ATCC No. CCL 61),HeLa (e.g., ATCC No. CCL 2), 293 (ATCC No. 1573; Graham et al., J. Gen.Virol. 36:59-72, 1977) and NS-1 cells. Other mammalian cell lines may beused within the present invention, including Rat Hep I (ATCC No. CRL1600), Rat Hep II (ATCC No. CRL 1548), TCMK (ATCC No. CCL 139), Humanlung (ATCC No. CCL 75.1), Human hepatoma (ATCC No. HTB-52), Hep G2 (ATCCNo. HB 8065), Mouse liver (ATCC No. CCL 29.1), NCTC 1469 (ATCC No. CCL9.1), SP2/0-Ag14 (ATCC No. 1581), HIT-T15 (ATCC No. CRL 1777), Jurkat(ATCC No. Tib 152) and RINm 5AHT₂B (Orskov and Nielson, FEBS229(1):175-178, 1988).

Mammalian expression vectors for use in carrying out the presentinvention will include a promoter capable of directing the transcriptionof a cloned gene or cDNA. Preferred promoters include viral promotersand cellular promoters. Viral promoters include the cytomegalovirusimmediate early promoter (Boshart et al., Cell 41:521-30, 1985),cytomegalovirus immediate late promoter, SV40 promoter (Subramani etal., Mol. Cell. Biol. 1:854-64, 1981), MMTV LTR, RSV LTR,metallothionein-1, adenovirus E1a. Cellular promoters include the mousemetallothionein-1 promoter (Palmiter et al., U.S. Pat. No. 4,579,821), amouse V_(κ) promoter (Bergman et al., Proc. Natl. Acad. Sci. USA81:7041-45, 1983; Grant et al., Nucl. Acids Res. 15:5496, 1987) and amouse V_(H) promoter (Loh et al., Cell 33:85-93, 1983). The choice ofpromoter will depend, at least in part, upon the level of expressiondesired or the recipient cell line to be transfected.

Such expression vectors may also contain a set of RNA splice siteslocated downstream from the promoter and upstream from the DNA sequenceencoding the peptide or protein of interest. Preferred RNA splice sitesmay be obtained from adenovirus and/or immunoglobulin genes. Alsocontained in the expression vectors is a polyadenylation signal locateddownstream of the coding sequence of interest. Suitable polyadenylationsignals include the early or late polyadenylation signals from SV40(Kaufman and Sharp, ibid.), the polyadenylation signal from theAdenovirus 5 E1B region and the human growth hormone gene terminator(DeNoto et al., Nuc. Acids Res. 9:3719-30, 1981). The expression vectorsmay include a noncoding viral leader sequence, such as the Adenovirus 2tripartite leader, located between the promoter and the RNA splicesites. Preferred vectors may also include enhancer sequences, such asthe SV40 enhancer. Expression vectors may also include sequencesencoding the adenovirus VA RNAs. Suitable expression vectors can beobtained from commercial sources (e.g., Stratagene, La Jolla, Calif.).

Vector constructs comprising cloned DNA sequences can be introduced intocultured mammalian cells by, for example, calcium phosphate-mediatedtransfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson,Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virology52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-45, 1982),or DEAE-dextran mediated transfection (Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley and Sons, Inc., N.Y., 1987).To identify cells that have stably integrated the cloned DNA, aselectable marker is generally introduced into the cells along with thegene or cDNA of interest. Preferred selectable markers for use incultured mammalian cells include genes that confer resistance to drugs,such as neomycin, hygromycin, and methotrexate. Other selectable markersinclude fluorescent proteins such as GFP (green fluorescent protein) orBFP (blue fluorescent protein). The selectable marker may be anamplifiable selectable marker. Preferred amplifiable selectable markersare the DHFR gene and the neomycin resistance gene. Selectable markersare reviewed by Thilly (Mammalian Cell Technology, ButterworthPublishers, Stoneham, Mass.).

Mammalian cells containing a suitable vector are allowed to grow for aperiod of time, typically 1-2 days, to begin expressing the DNAsequence(s) of interest. Drug selection is then applied to select forgrowth of cells that are expressing the selectable marker in a stablefashion. For cells that have been transfected with an amplifiable,selectable marker the drug concentration may be increased in a stepwisemanner to select for increased copy number of the cloned sequences,thereby increasing expression levels. Cells expressing the introducedsequences are selected and screened for production of the protein ofinterest in the desired form or at the desired level. Cells that satisfythese criteria can then be cloned and scaled up for production. Cellsmay also be selected for transfection based on their expression of GFPby sorting for GFP-positive cells using a flow cytometer.

Protocols for the transfection of mammalian cells are well known tothose of ordinary skill in the art. Representative methods includecalcium phosphate mediated transfection, electroporation, lipofection,retroviral, adenoviral and protoplast fusion-mediated transfection (seeSambrook et al., supra). Naked vector constructs can also be taken up bymuscle cells or other suitable cells subsequent to injection into themuscle of a mammal (or other animals).

Numerous insect host cells known in the art can also be useful withinthe present invention, in light of the subject specification. Forexample, the use of baculoviruses as vectors for expressing heterologousDNA sequences in insect cells has been reviewed by Atkinson et al.(Pestic. Sci. 28:215-24, 1990).

Numerous plant host cells known in the art can also be useful within thepresent invention, in light of the subject specification. For example,the use of Agrobacterium rhizogenes as vectors for expressing genes inplant cells has been reviewed by Sinkar et al. (J. Biosci. (Bangalore)11:47-58, 1987).

Within related aspects of the present invention, proteins of the presentinvention, may be expressed in a transgenic animal whose germ cells andsomatic cells contain a gene which encodes the desired protein and whichis operably linked to a promoter effective for the expression of thegene. Alternatively, in a similar manner transgenic animals may beprepared that lack the desired gene (e.g., “knockout” mice). Suchtransgenics may be prepared in a variety non-human animals, includingmice, rats, rabbits, sheep, dogs, goats and pigs (see Hammer et al.,Nature 315:680-83, 1985, Palmiter et al., Science 222:809-14, 1983,Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-42, 1985, Palmiterand Brinster, Cell 41:343-45, 1985, and U.S. Pat. Nos. 5,175,383,5,087,571, 4,736,866, 5,387,742, 5,347,075, 5,221,778, and 5,175,384).Briefly, an expression vector, including a nucleic acid molecule to beexpressed together with appropriately positioned expression controlsequences, is introduced into pronuclei of fertilized eggs, for example,by microinjection. Integration of the injected DNA is detected by blotanalysis of DNA from tissue samples. It is preferred that the introducedDNA be incorporated into the germ line of the animal so that it ispassed on to the animal's progeny. Tissue-specific expression may beachieved through the use of a tissue-specific promoter, or through theuse of an inducible promoter, such as the metallothionein gene promoter(Palmiter et al., 1983, ibid), which allows regulated expression of thetransgene.

Animals which produce mutant forms of Fkh^(sf) other than the naturallyoccurring scurfy mutant (“sf”), or in genetic backgrounds different fromthe naturally occurring mutant, may be readily produced given thedisclosure provided herein.

Proteins can be isolated by, among other methods, culturing suitablehost and vector systems to produce the recombinant translation productsof the present invention. Supernatants from such cell lines, or proteininclusions or whole cells where the protein is not excreted into thesupernatant, can then be treated by a variety of purification proceduresin order to isolate the desired proteins. For example, the supernatantmay be first concentrated using commercially available proteinconcentration filters, such as an Amicon or Millipore Pelliconultrafiltration unit. Following concentration, the concentrate may beapplied to a suitable purification matrix such as, for example, ananti-protein antibody bound to a suitable support. Alternatively, anionor cation exchange resins may be employed in order to purify theprotein. As a further alternative, one or more reverse-phase highperformance liquid chromatography (RP-HPLC) steps may be employed tofurther purify the protein. Other methods of isolating the proteins ofthe present invention are well known in the skill of the art.

A protein is deemed to be “isolated” within the context of the presentinvention if no other (undesired) protein is detected pursuant toSDS-PAGE analysis followed by Coomassie blue staining. Within otherembodiments, the desired protein can be isolated such that no other(undesired) protein is detected pursuant to SDS-PAGE analysis followedby silver staining.

Assays for Selecting Molecules which Modulate the Immune System

As noted above, the present invention provides methods for selectingand/or isolating molecules which are capable of modulating the immunesystem. Representative examples of suitable assays include the yeast andmammalian 2-hybrid systems (e.g., Dang et al., Mol. Cell. Biol. 11:954,1991; Fearon et al., Proc. Natl. Acad. Sci. USA 89:7958, 1992), DNAbinding assays, antisense assays, traditional protein binding assays(e.g., utilizing ¹²⁵I or time-resolved fluorescence),immunoprecipitation coupled with gel electrophoresis and direct proteinsequencing, transcriptional analysis of Fkh^(sf) regulated genes,cytokine production and proliferation assays.

For example, within one embodiment proteins that directly interact withFkh^(sf) can be detected by an assay such as a yeast 2-hybrid bindingsystem (see, e.g., U.S. Pat. Nos. 5,283,173, 5,468,614, 5,610,015, and5,667,973). Briefly, in a two-hybrid system, a fusion of a DNA-bindingdomain-Fkh^(sf) protein (e.g., GAL4-Fkh^(sf) fusion) is constructed andtransfected into a cell containing a GAL4 binding site linked to aselectable marker gene. The whole Fkh^(sf) protein or subregions ofFkh^(sf) may be used. A library of cDNAs fused to the GAL4 activationdomain is also constructed and co-transfected. When the cDNA in thecDNA-GAL4 activation domain fusion encodes a protein that interacts withFkh^(sf), the selectable marker is expressed. Cells containing the cDNAare then grown, the construct isolated and characterized. Other assaysmay also be used to identify interacting proteins. Such assays includeELISA, Western blotting, co-immunoprecipitations, in vitrotranscription/translation analysis and the like.

Within another aspect of the present invention, methods are provided fordetermining whether a selected molecule is capable of modulating theimmune system, comprising the steps of (a) exposing a selected candidatemolecule to cells which express Fkh^(sf), or, mutant Fkh^(sf), and (b)determining whether the molecule modulates the activity of Fkh^(sf), andthereby determining whether said molecule can modulate the immunesystem. Cells for such tests may derive from (a) normal lymphocytes, (b)cell lines engineered to overexpress the FKH^(sf) (or Fkh^(sf)) protein(or mutant forms thereof) or (c) transgenic animals engineered toexpress said protein. Cells from such transgenic mice are characterized,in part, by a hyporesponsive state including diminished cell number anda decreased responsiveness to various stimuli (e.g., Example 8).

It should be noted that while the methods recited herein may refer tothe analysis of an individual test molecule, that the present inventionshould not be so limited. In particular, the selected molecule may becontained within a mixture of compounds. Hence, the recited methods mayfurther comprise the step of isolating the desired molecule.Furthermore, it should be understood that candidate molecules can beassessed for their ability to modulate the immune system by a number ofparameters, including for example, T-cell proliferation, cytokineproduction, and the like.

Candidate Molecules

A wide variety of molecules may be assayed for their ability to modulatethe immune system. Representative examples which are discussed in moredetail below include organic molecules, proteins or peptides, andnucleic acid molecules.

1. Organic Molecules

Numerous organic molecules may be assayed for their ability to modulatethe immune system. For example, within one embodiment of the inventionsuitable organic molecules may be selected either from a chemicallibrary, wherein chemicals are assayed individually, or fromcombinatorial chemical libraries where multiple compounds are assayed atonce, then deconvoluted to determine and isolate the most activecompounds.

Representative examples of such combinatorial chemical libraries includethose described by Agrafiotis et al., “System and method ofautomatically generating chemical compounds with desired properties,”U.S. Pat. No. 5,463,564; Armstrong, R. W., “Synthesis of combinatorialarrays of organic compounds through the use of multiple componentcombinatorial array syntheses,” WO 95/02566; Baldwin, J. J. et al.,“Sulfonamide derivatives and their use,” WO 95/24186; Baldwin, J. J. etal., “Combinatorial dihydrobenzopyran library,” WO 95/30642; Brenner,S., “New kit for preparing combinatorial libraries,” WO 95/16918;Chenera, B. et al., “Preparation of library of resin-bound aromaticcarbocyclic compounds,” WO 95/16712; Ellman, J. A., “Solid phase andcombinatorial synthesis of benzodiazepine compounds on a solid support,”U.S. Pat. No. 5,288,514; Felder, E. et al., “Novel combinatorialcompound libraries,” WO 95/16209; Lerner, R. et al., “Encodedcombinatorial chemical libraries,” WO 93/20242; Pavia, M. R. et al., “Amethod for preparing and selecting pharmaceutically useful non-peptidecompounds from a structurally diverse universal library,” WO 95/04277;Summerton, J. E. and D. D. Weller, “Morpholino-subunit combinatoriallibrary and method,” U.S. Pat. No. 5,506,337; Holmes, C., “Methods forthe Solid Phase Synthesis of Thiazolidinones, Metathiazanones, andDerivatives thereof,” WO 96/00148; Phillips, G. B. and G. P. Wei,“Solid-phase Synthesis of Benzimidazoles,” Tet. Letters 37:4887-90,1996; Ruhland, B. et al., “Solid-supported Combinatorial Synthesis ofStructurally Diverse β-Lactams,” J. Amer. Chem. Soc. 111:253-54, 1996;Look, G. C. et al., “The Indentification of Cyclooxygenase-1 Inhibitorsfrom 4-Thiazolidinone Combinatorial Libraries,” Bioorg and Med. Chem.Letters 6:707-12, 1996.

2. Proteins and Peptides

A wide range of proteins and peptides make likewise be utilized ascandidate molecules for modulating the immune system.

a. Combinatorial Peptide Libraries

Peptide molecules which modulate the immune system may be obtainedthrough the screening of combinatorial peptide libraries. Such librariesmay either be prepared by one of skill in the art (see, e.g., U.S. Pat.Nos. 4,528,266 and 4,359,535, and Patent Cooperation Treaty PublicationNos. WO 92/15679, WO 92/15677, WO 90/07862, WO 90/02809), or purchasedfrom commercially available sources (e.g., New England Biolabs™ PhageDisplay Peptide Library Kit).

b. Antibodies

Antibodies which modulate the immune system may readily be preparedgiven the disclosure provided herein. Within the context of the presentinvention, antibodies are understood to include monoclonal antibodies,polyclonal antibodies, anti-idiotypic antibodies, antibody fragments(e.g., Fab, and F(ab′)₂, F_(v) variable regions, or complementaritydetermining regions). As discussed above, antibodies are understood tobe specific against Fkh^(sf) if they bind with a K_(a) of greater thanor equal to 10⁷M, preferably greater than of equal to 10⁸M. The affinityof a monoclonal antibody or binding partner, as well as inhibition ofbinding can be readily determined by one of ordinary skill in the art(see Scatchard, Ann. N.Y. Acad. Sci. 51:660-72, 1949).

Briefly, polyclonal antibodies may be readily generated by one ofordinary skill in the art from a variety of warm-blooded animals such ashorses, cows, various fowl, rabbits, mice, or rats. Typically, Fkh^(sf),or a unique peptide thereof of 13-20 amino acids (preferably conjugatedto keyhole limpet hemocyanin by cross-linking with glutaraldehyde) isutilized to immunize the animal through intraperitoneal, intramuscular,intraocular, or subcutaneous injections, in conjunction with an adjuvantsuch as Freund's complete or incomplete adjuvant. Following severalbooster immunizations, samples of serum are collected and tested forreactivity to the protein or peptide. Particularly preferred polyclonalantisera will give a signal on one of these assays that is at leastthree times greater than background. Once the titer of the animal hasreached a plateau in terms of its reactivity to the protein, largerquantities of antisera may be readily obtained either by weeklybleedings, or by exsanguinating the animal.

Monoclonal antibodies may also be readily generated using conventionaltechniques (see U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and4,411,993 which are incorporated herein by reference; see alsoMonoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, andAntibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold SpringHarbor Laboratory Press, 1988).

Other techniques may also be utilized to construct monoclonal antibodies(see William D. Huse et al., “Generation of a Large CombinationalLibrary of the Immunoglobulin Repertoire in Phage Lambda,” Science246:1275-81, December 1989; see also L. Sastry et al., “Cloning of theImmunological Repertoire in Escherichia coli for Generation ofMonoclonal Catalytic Antibodies: Construction of a Heavy Chain VariableRegion-Specific cDNA Library,” Proc. Natl. Acad. Sci. USA 86:5728-32,August 1989; see also Michelle Alting-Mees et al., “Monoclonal AntibodyExpression Libraries: A Rapid Alternative to Hybridomas,” Strategies inMolecular Biology 3:1-9, January 1990).

A wide variety of assays may be utilized to determine the presence ofantibodies which are reactive against the Fkh^(sf) (or the mutant formsof Fkh^(sf) described herein), including for example countercurrentimmuno-electrophoresis, radioimmunoassays, radioimmunoprecipitations,enzyme-linked immuno-sorbent assays (ELISA), dot blot assays, westernblots, immunoprecipitation, Inhibition or Competition Assays, andsandwich assays (see U.S. Pat. Nos. 4,376,110 and 4,486,530; see alsoHarlow and Lane (eds.), Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, 1988).

Once suitable antibodies have been obtained, they may be isolated orpurified by many techniques well known to those of ordinary skill in theart (see Harlow and Lane (eds.), Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, 1988). Suitable techniques includepeptide or protein affinity columns, HPLC or RP-HPLC, purification onprotein A or protein G columns, or any combination of these techniques.

Antibodies of the present invention may be utilized not only formodulating the immune system, but for diagnostic tests (e.g., todetermine the presence of an FKH^(sf) or Fkh^(sf) protein or peptide),for therapeutic purpose, or for purification of proteins.

c. Mutant Fkh^(sf)

As described herein and below in the Examples, altered versions ofFkh^(sf), may be utilized to inhibit the normal activity of Fkh^(sf),thereby modulating the immune system (see generally, nucleic acidmolecules and proteins above).

Further mutant or altered forms of FKH^(sf) or Fkh^(sf) may be utilizedfor a wide variety of in vitro assays (e.g., in order to examine theeffect of such proteins in various models), or, for the development ofantibodies.

3. Nucleic Acid Molecules

Within other aspects of the invention, nucleic acid molecules areprovided which are capable of modulating the immune system. For example,within one embodiment antisense oligonucleotide molecules are providedwhich specifically inhibit expression of FKH^(sf) or Fkh^(sf) nucleicacid sequences, or, of mutant FKH^(sf) or Fkh^(sf) (see generally,Hirashima et al., in Molecular Biology of RNA: New Perspectives (M.Inouye and B. S. Dudock, eds., 1987 Academic Press, San Diego, p. 401);Oligonucleotides: Antisense Inhibitors of Gene Expression (J. S. Cohen,ed., 1989 MacMillan Press, London); Stein and Cheng, Science261:1004-12, 1993; WO 95/10607; U.S. Pat. No. 5,359,051; WO 92/06693;and EP-A2-612844). Briefly, such molecules are constructed such thatthey are complementary to, and able to form Watson-Crick base pairswith, a region of transcribed Fkh^(sf) mRNA sequence. The resultantdouble-stranded nucleic acid interferes with subsequent processing ofthe mRNA, thereby preventing protein synthesis.

Within other aspects of the invention, ribozymes are provided which arecapable of inhibiting FKH^(sf) or Fkh^(sf), or mutant forms FKH^(sf) orFkh^(sf). As used herein, “ribozymes” are intended to include RNAmolecules that contain anti-sense sequences for specific recognition,and an RNA-cleaving enzymatic activity. The catalytic strand cleaves aspecific site in a target RNA at greater than stoichiometricconcentration. A wide variety of ribozymes may be utilized within thecontext of the present invention, including for example, the hammerheadribozyme (for example, as described by Forster and Symons, Cell48:211-20, 1987; Haseloff and Gerlach, Nature 328:596-600, 1988; Walbotand Bruening, Nature 334:196, 1988; Haseloff and Gerlach, Nature334:585, 1988); the hairpin ribozyme (for example, as described byHaselhoff et al., U.S. Pat. No. 5,254,678, issued Oct. 19, 1993 andHempel et al., European Patent Publication No. 0 360 257, published Mar.26, 1990); and Tetrahymena ribosomal RNA-based ribozymes (see Cech etal., U.S. Pat. No. 4,987,071). Ribozymes of the present inventiontypically consist of RNA, but may also be composed of DNA, nucleic acidanalogs (e.g., phosphorothioates), or chimerics thereof (e.g.,DNA/RNA/RNA).

4. Labels

FKH^(sf) or Fkh^(sf), (as well as mutant forms thereof), or, any of thecandidate molecules described above and below, may be labeled with avariety of compounds, including for example, fluorescent molecules,toxins, and radionuclides. Representative examples of fluorescentmolecules include fluorescein, Phycobili proteins, such asphycoerythrin, rhodamine, Texas red and luciferase. Representativeexamples of toxins include ricin, abrin diphtheria toxin, cholera toxin,gelonin, pokeweed antiviral protein, tritin, Shigella toxin, andPseudomonas exotoxin A. Representative examples of radionuclides includeCu-64, Ga-67, Ga-68, Zr-89, Ru-97, Tc-99m, Rh-105, Pd-109, In-111,I-123, I-125, I-131, Re-186, Re-188, Au-198, Au-199, Pb-203, At-211,Pb-212 and Bi-212. In addition, the antibodies described above may alsobe labeled or conjugated to one partner of a ligand binding pair.Representative examples include avidin-biotin, and riboflavin-riboflavinbinding protein.

Methods for conjugating or labeling the molecules described herein withthe representative labels set forth above may be readily accomplished byone of ordinary skill in the art (see Trichothecene Antibody Conjugate,U.S. Pat. No. 4,744,981; Antibody Conjugate, U.S. Pat. No. 5,106,951;Fluorogenic Materials and Labeling Techniques, U.S. Pat. No. 4,018,884;Metal Radionuclide Labeled Proteins for Diagnosis and Therapy, U.S. Pat.No. 4,897,255; and Metal Radionuclide Chelating Compounds for ImprovedChelation Kinetics, U.S. Pat. No. 4,988,496; see also Inman, Jakoby andWilchek (eds.), Methods In Enzymology, Vol. 34, Affinity Techniques,Enzyme Purification: Part B, Academic Press, New York, 1974, p. 30; seealso Wilchek and Bayer, “The Avidin-Biotin Complex in BioanalyticalApplications,” Anal. Biochem. 171:1-32, 1988).

Pharmaceutical Compositions

As noted above, the present invention also provides a variety ofpharmaceutical compositions, comprising one of the above-describedmolecules which modulates the immune system, along with apharmaceutically or physiologically acceptable carrier, excipients ordiluents. Generally, such carriers should be nontoxic to recipients atthe dosages and concentrations employed. Ordinarily, the preparation ofsuch compositions entails combining the therapeutic agent with buffers,antioxidants such as ascorbic acid, low molecular weight (less thanabout 10 residues) polypeptides, proteins, amino acids, carbohydratesincluding glucose, sucrose or dextrins, chelating agents such as EDTA,glutathione and other stabilizers and excipients. Neutral bufferedsaline or saline mixed with nonspecific serum albumin are exemplaryappropriate diluents. Preferably, the pharmaceutical composition (or,‘medicament’) is provided in sterile, pyrogen-free form.

In addition, the pharmaceutical compositions of the present inventionmay be prepared for administration by a variety of different routes. Inaddition, pharmaceutical compositions of the present invention may beplaced within containers, along with packaging material which providesinstructions regarding the use of such pharmaceutical compositions.Generally, such instructions will include a tangible expressiondescribing the reagent concentration, as well as within certainembodiments, relative amounts of excipient ingredients or diluents(e.g., water, saline or PBS) which may be necessary to reconstitute thepharmaceutical composition.

Methods of Treatment

The present invention also provides methods for modulating the immunesystem. Through use of the molecules described herein which modulate theimmune system, a wide variety of conditions in warm blooded animals maybe readily treated or prevented. Examples of warm-blooded animals thatmay be treated include both vertebrates and mammals, including forexample humans, horses, cows, pigs, sheep, dogs, cats, rats and mice.Such methods may have therapeutic value in patients with altered immunesystems. This would include such patients as those undergoingchemotherapy of those with various immunodeficiency syndromes, as wellas patients with a T cell mediated autoimmune disease. Therapeutic valuemay also be recognized from utility as a vaccine adjuvant.

Therapeutic molecules, depending on the type of molecule, may beadministered via a variety of routes in a variety of formulations. Forexample, within one embodiment organic molecules may be delivered byoral or nasal routes, or by injection (e.g., intramuscularly,intravenously, and the like).

Within one aspect, methods are provided for modulating the immunesystem, comprising the step of introducing into lymphoid cells a vectorwhich directs the expression of a molecule which modulates the immunesystem, and administering the vector containing cells to a warm-bloodedanimal. Within other related embodiments, the vector may be directlyadministered to a desired target location (e.g., the bone marrow).

A wide variety of vectors may be utilized for such therapeutic purposes,including both viral and non-viral vectors. Representative examples ofsuitable viral vectors include herpes viral vectors (e.g., U.S. Pat. No.5,288,641), adenoviral vectors (e.g., WO 94/26914, WO 93/9191 WO99/20778; WO 99/20773; WO 99/20779; Kolls et al., PNAS 91(1):215-19,1994; Kass-Eisler et al., PNAS 90(24):11498-502, 1993; Guzman et al.,Circulation 88(6):2838-48, 1993; Guzman et al., Cir. Res. 73(6):1202-07,1993; Zabner et al., Cell 75(2):207-16, 1993; Li et al., Hum Gene Ther.4(4):403-09, 1993; Caillaud et al., Eur. J. Neurosci. 5(10):1287-91,1993; Vincent et al., Nat. Genet. 5(2):130-34, 1993; Jaffe et al., Nat.Genet. 1(5):372-78, 1992; and Levrero et al., Gene 101(2):195-202,1991), adeno-associated viral vectors (WO 95/13365; Flotte et al., PNAS90(22):10613-617, 1993), baculovirus vectors, parvovirus vectors(Koering et al., Hum. Gene Therap. 5:457-63, 1994), pox virus vectors(Panicali and Paoletti, PNAS 79:4927-31, 1982; and Ozaki et al.,Biochem. Biophys. Res. Comm. 193(2):653-60, 1993), and retroviruses(e.g., EP 0,415,731; WO 90/07936; WO 91/0285, WO 94/03622; WO 93/25698;WO 93/25234; U.S. Pat. No. 5,219,740; WO 93/11230; WO 93/10218). Viralvectors may likewise be constructed which contain a mixture of differentelements (e.g., promoters, envelope sequences and the like) fromdifferent viruses, or non-viral sources. Within various embodiments,either the viral vector itself, or a viral particle which contains theviral vector may be utilized in the methods and compositions describedbelow.

Within other embodiments of the invention, nucleic acid molecules whichencode a molecule which modulates the immune system (e.g., a mutantFkh^(sf), or, an antisense or ribozyme molecule which cleaves Fkh^(sf))may be administered by a variety of alternative techniques, includingfor example administration of asialoosomucoid (ASOR) conjugated withpoly-L-lysine DNA complexes (Cristano et al., PNAS 92122-126, 1993), DNAlinked to killed adenovirus (Curiel et al., Hum. Gene Ther. 3(2):147-54,1992), cytofectin-mediated introduction (DMRIE-DOPE, Vical, California),direct DNA injection (Acsadi et al., Nature 352:815-18, 1991); DNAligand (Wu et al., J. of Biol. Chem. 264:16985-987, 1989); lipofection(Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-17, 1989); liposomes(Pickering et al., Circ. 89(1):13-21, 1994; and Wang et al., PNAS84:7851-55, 1987); microprojectile bombardment (Williams et al., PNAS88:2726-30, 1991); and direct delivery of nucleic acids which encode theprotein itself either alone (Vile and Hart, Cancer Res. 53: 3860-64,1993), or utilizing PEG-nucleic acid complexes.

Representative examples of molecules which may be expressed by thevectors of present invention include ribozymes and antisense molecules,each of which are discussed in more detail above.

As will be evident to one of skill in the art, the amount and frequencyof administration will depend, of course, on such factors as the natureand severity of the indication being treated, the desired response, thecondition of the patient, and so forth. Typically, the compositions maybe administered by a variety of techniques, as noted above.

The following examples are offered by way of illustration, and not byway of limitation.

EXAMPLES Example 1 Identification of the Gene Responsible for the ScurfyMutant A. Cloning of a Scurfy Gene

The original scurfy mutation arose spontaneously in the partially inbredMR stock at Oak Ridge National Laboratory (ORNL) in 1949. Backcrossanalysis was used to fine map the peri-centromeric region of the Xchromosome containing the mouse Scurfy mutation. A physical map coveringthe same region was generated concurrently through the isolation ofoverlapping yeast and bacterial artificial chromosomes (YACs and BACs).Once the candidate region was narrowed down to ˜500 kilobase pairs (kb),large-scale DNA sequencing was performed on 4 overlapping BAC clones.All the transcription units in this 500 kb region were identifiedthrough a combination of sequence database searching and the applicationof computer exon prediction programs. Candidate genes were then screenedfor Scurfy-specific mutations by comparing the sequences of cDNAsobtained by the Reverse Transcription-Polymerase Chain Reaction (RT-PCR)procedure from normal and Scurfy-derived RNA samples. In one gene,referred to here as Fkh^(sf), a two base pair (bp) insertion was foundin the coding region of the Scurfy cDNA, relative to the normal cDNA.The insertion was confirmed by comparing the DNA sequences of PCRproducts derived from the genomic DNA of several mouse strains,including the Scurfy mutant. Again, the two bp insertion was found onlyin the Scurfy sample, establishing this as the probable cause of theScurfy defect.

The mouse Fkh^(sf) gene is contained within the BAC clone 8C22, and hasbeen completely sequenced. It spans ˜14 kb and contains 11 coding exons.The locations of exon breaks were initially identified by computeranalysis of the genomic DNA sequence, using the GenScan exon predictionprogram; exon locations were then confirmed by direct comparison of cDNAsequences derived from normal mouse tissues to the genomic sequence.

The length of cDNA obtained is 2160 bp; the coding region spans 1287 bpof that, encoding a protein of 429 amino acids. FIG. 1 shows thenucleotide sequence of the mouse Fkh^(sf) cDNA; translation is predictedto initiate at position 259 and terminate at position 1546. FIG. 2 showsthe amino acid sequence of mouse Fkh^(sf).

B. Generation of Fkh^(sf) Transgenic Mice.

The identity of the Fkh^(sf) gene as the true cause of the Scurfyphenotype was confirmed in transgenic mice. Briefly, a 30 kb fragment ofthe normal genomic DNA, including the ˜7 kb coding region of theFkh^(sf) gene, as well as ˜20 kb of upstream flanking sequences and ˜4kb of downstream sequences (FIG. 5) was microinjected into normal mouseone-cell embryos. Five individual founder animals were generated, eachwith distinct integrations, and a male animal from each transgenic linewas crossed to a female sf carriers. Male offspring carrying both thetransgene (normal Fkh^(sf)) and sf mutation (mutant Fkh^(sf)) wereanalyzed.

Analysis consisted of examination of animals for runting, scaly skin,fur abnormalities and other hallmarks of the scurfy phenotype. Inaddition, lymphoid tissues (thymus, spleen and nodes) were harvested andtheir size and cell number examined and compared to both normal animalsas well as scurfy mice. For all five transgenic lines, male sf progenythat carried the transgene were normal in size and weight and appearedhealthy in all respects. Lymph node size in these transgenic mice wassimilar to (or smaller than) that of normal animals (FIG. 6) and therewas no sign of activated T cells. These parameters are extremelydifferent from sf mice and indicate that addition of the normal Fkh^(sf)gene can overcome the defect found in scurfy mice, thus confirming thatthe mutation in the Fkh^(sf) gene is the cause of Scurfy disease.

Example 2 Generation of FKH^(sf) cDNA

A complementary DNA (cDNA) encoding the complete mouse Fkh^(sf) proteinmay be obtained by a reverse-transcriptase polymerase chain reaction(RT-PCR) procedure. More specifically, first-strand cDNA is generated byoligo dT priming 5 ug of total RNA from a suitable source (eg., mousespleen) and extending with reverse transcriptase under standardconditions (eg., Gibco/BRL SuperScript kit). An aliquot of thefirst-strand cDNA is then subjected to 35 cycles of PCR (94° C. for 30sec, 63° C. for 30 sec, 72° C. for 2 min) in the presence of the forwardand reverse primers (Forward primer: GCAGATCTCC TGACTCTGCC TTC; Reverseprimer: GCAGATCTGA CAAGCTGTGT CTG) (0.2 mM final concentration), 60 mMTris-HCl, 15 mM ammonium sulfate, 1.5 mM magnesium chloride, 0.2 mM eachdNTP and 1 unit of Taq polymerase.

Example 3 Generation of the Human Ortholog to Murine Fkh^(sf)

A human FKH^(sf) cDNA encoding the complete FKH^(sf) protein may beobtained by essentially the same procedure as described in Example 2. Inparticular, starting with total spleen RNA, and utilizing the followingoligonucleotide primers (Forward primer: AGCCTGCCCT TGGACAAGGA C;Reverse primer: GCAAGACAGT GGAAACCTCA C), and the same PCR conditionsoutlined above, except with a 60° C. annealing temperature.

FIG. 3 shows the nucleotide sequence of the 1869 bp cDNA obtained todate (including an 1293 bp coding region); translation is predicted toinitiate at position 189 and terminate at position 1482. FIG. 4 showsthe sequence of the 431 amino acid human FKH^(sf) protein. Comparison ofthe predicted coding region of the human gene to the mouse cDNA sequencereveals nearly identical exon structure and 86.1% amino acid sequenceidentity across the entire protein.

Example 4 Methods for Detecting Scurfy Mutations

As noted above, the Scurfy mutation was originally discovered bydirectly sequencing cDNAs derived by RT-PCR of sf and normal mouse RNAsamples, and confirmed by sequencing the same region from genomic DNA.The nature of the mutation (i.e., a 2 bp insertion) lends itself to anumber of different mutation detection assays. The first is based ondifferential hybridization of oligonucleotide probes. Such ahybridization-based assay could allow quantitative analysis ofallele-specific expression.

As an example, a 360 bp DNA fragment is amplified from 1^(st) strandcDNA using the following oligos:

DMO5985 (forward): CTACCCACTGCTGGCAAATG (ntd. 825-844 of FIG. 1) DMO6724(reverse): GAAGGAACTATTGCCATGGCTTC (ntd. 1221-1199)

The PCR products are run on an 1.8% agarose gel, transferred to nylonmembrane and probed with end-labeled oligonucleotides that arecomplementary to the region corresponding to the site of theScurfy-specific 2 bp insertion. Two separate hybridization reactions areperformed to detect the normal and Scurfy PCR products, using theoligonucleotides below (the site of the 2 bp insertion is shown inbold):

Normal: ATGCAGCAAGAGCTCTTGTCCATTGAGG DMO7439 Scurfy:GCAGCAAGAGCTCTTTTGTCCATTGAGG DMO6919

The Scurfy mutation can also be detected by a cold Single-StrandConformation Polymorphism (cSSCP) assay. In this assay, the same PCRproducts described above are run on 20% acrylamide (TBE) gels afterstrand denaturation. The Scurfy insertion causes a shift in strandmobility, relative to the normal sequence, and the separate strands aredetected after staining with ethidium bromide.

Example 5 Fkh^(sf) Gene Expression

Semi-quantitative RT-PCR has been used to analyze the pattern of mouseand human Fkh^(sf) gene expression in a wide variety of tissues and celllines. Levels of expression are normalized to the ubiquitously expressedDAD-1 gene. In short, the Fkh^(sf) gene is expressed, albeit at very lowlevels, in nearly every tissue examined thus far, including thymus,spleen, sorted CD4+ and CD4−CD8− T-lymphocytes, as well as kidney,brain, and various mouse and human T-cell lines and human tumors.Absence of expression, however, was noted in freshly sorted mouseB-cells.

As expected, no differences in level of expression were observed innormal vs. Scurfy tissues in the RT-PCR assays.

Example 6 In Vitro Expression of Fkh^(sf)

Full-length mouse and human Fkh^(sf) cDNAs, as well as varioussub-regions of the cDNAs are cloned into vectors which allow expressionin mammalian cells (such as the human Jurkat T-cell line), E. coli oryeast. The E. coli or yeast systems can be used for production ofprotein for the purpose of raising Fkh^(sf)-specific antibodies (seebelow).

Example 7 Generation of Anti-Fkh^(sf) Antibodies

Protein expressed from vectors described in Example 6 are used toimmunize appropriate animals for the production of FKH^(sf) specificantibodies. Either full length or truncated proteins can be used forthis purpose. Protein can be obtained, for example, from bacteria suchas E. coli, insect cells or mammalian cells. Animal species can includemouse, rabbit, guinea pig, chicken or other. Rabbit antisera specificfor FKH^(sf) has been generated, as determined by biochemicalcharacterization (immunoprecipitation and western blotting).

Example 8 Assay for Function of an FKH^(sf) Gene

Since loss of function of the FKH^(sf) protein results in the phenotypeobserved in scurfy animals (wasting, hyperactive immune responsivenessand death), assays are described for assessing excessive expression ofthe FKH^(sf) protein. Transgenic animals (described in Example 1) areexamined for their state of immune competence, using several differentparameters. Animals are examined for the number of lymphoid cellspresent in lymph nodes and thymus (FIG. 7) as well as the responsivenessof T cells to in vitro stimulation (FIG. 8).

Scurfy mutant animals have roughly twice as many cells in their lymphnodes as normal animals, whereas mice which express excess levels of thenormal FKH^(sf) protein contain roughly one-third as many cells (FIG.7). Further, the number of thymocytes is normal (FIG. 7) as is theircell surface phenotype as assessed by flow cytometry using standardantisera (not shown), indicating that there is no developmental defectassociated with excess FKH^(sf) protein.

Normal, scurfy and transgenic animals are further examined for theirproliferative responses to T cell stimulation. CD4+ T cells are reactedwith antibodies to CD3 and CD28 and their proliferative responsemeasured using radioactive thymidine incorporation. Whereas only scurfycells divide in the absence of stimulation, normal cells respond wellfollowing stimulation. FKH^(sf) transgenic cells also respond tostimulation, however the response is significantly less than that ofnormal cells (FIG. 8). This indicates that CD4+ T cells that expressexcess FKH^(sf) have a diminished capacity to respond to stimuli.

Example 9 Human FKH^(sf) cDNA Sequence is Related to JM2

A modified version of the human FKH^(sf) cDNA sequence exists in theGenBank public sequence database. This sequence is called JM2 (GenBankacc. # AJ005891), and is the result of the application of exonprediction programs to the genomic sequence containing the FKH^(sf) gene(Strom, T. M. et al., unpublished—see GenBank acc. # AJ005891). Incontrast, the structure of the FKH^(sf) cDNA was determinedexperimentally. The GAP program of the Genetics Computer Group (GCG;Madison, USA) Wisconsin sequence analysis package was used to comparethe two sequences, and the differences are illustrated in FIG. 9. The 5′ends of the two sequences differ in their location within the context ofthe genomic DNA sequence, the second coding exon of FKH^(sf) is omittedfrom JM2, and the last intron of the FKH^(sf) gene is unspliced in theJM2 sequence. These differences result in a JM2 protein with a shorteramino-terminal domain, relative to FKH^(sf), and a large insertionwithin the forkhead domain (see below) at the carboxy-terminus.

Example 10 The FKH^(sf) Protein is Conserved Across Species

The FKH^(sf) protein can be divided into sub-regions, based on sequencemotifs that may indicate functional domains. The two principal motifs inFKH^(sf) are the single zinc finger (ZNF) of the C₂H₂ class in themiddle portion of the protein, and the forkhead, or winged-helix domainat the extreme carboxy-terminus of the protein. For the purposes ofcharacterizing the degree of homology between FKH^(sf) and otherproteins, we have split the protein up into four regions:

-   -   Amino-terminal domain: residues 1-197 of FIG. 2 residues 1-198        of FIG. 4    -   Zinc finger domain: residues 198-221 of FIG. 2 residues 199-222        of FIG. 4    -   Middle domain: residues 222-336 of FIG. 2 residues 223-336 of        FIG. 4    -   Forkhead domain: residues 337-429 of FIG. 2 residues 337-431 of        FIG. 4

Using the Multiple Sequence Alignment program from the DNAStar sequenceanalysis package, the Lipman-Pearson algorithm was employed to determinethe degree of similarity between the human FKH^(sf) and mouse Fkh^(sf)proteins across these four domains. The results are shown in FIG. 10.The similarity indices ranged from 82.8% to 96.4%, indicating that thisprotein is very highly conserved across species.

Example 11 Identification of Novel Fkh^(sf)-Related Genes

The unique features of the FKH^(sf) gene sequence may be used toidentify other novel genes (and proteins) which fall into the samesub-class of forkhead-containing molecules. The FKH^(sf) protein isunique in its having a single zinc finger domain amino-terminal to theforkhead domain as well as in the extreme carboxy-terminal position ofthe forkhead domain. A degenerate PCR approach may be taken to isolatenovel genes containing a zinc finger sequence upstream of a forkheaddomain. By way of example, the following degenerate primers weresynthesized (positions of degeneracy are indicated by parentheses, and“I” indicates the nucleoside inosine):

Forward primer: CA(TC)GGIGA(GA)TG(CT)AA(GA)TGG Reverse primer:(GA)AACCA(GA)TT(AG)TA(AGT)AT(CT)TC(GA)TT

The forward primer corresponds to a region within the zinc fingersequence and the reverse primer corresponds to a region in the middle ofthe forkhead domain. These primers were used to amplify first-strandcDNA produced as in Example 2 from a variety of human tissues (includingliver, spleen, brain, lung, kidney, etc.). The following PCR conditionswere used: forward and reverse primers at 0.2 mM final concentration, 60mM Tris-HCl, 15 mM ammonium sulfate, 1.5 mM magnesium chloride, 0.2 mMeach dNTP and 1 unit of Taq polymerase, subjected to 35 cycles (94° C.for 30 sec, 50° C. for 30 sec, 72° C. for 2 min). PCR products werevisualized on a 1.8% agarose gel (run in 1×TAE) and sub-cloned into theTA cloning vector (Invitrogen, Carlsbad, Calif.); individual clones weresequenced and used for further characterization of full-length cDNAs.

Alternatively, the unique regions of the FKH^(sf) gene (i.e., the“Amino-terminal” and “Middle” domains) may be used to screen cDNAlibraries by hybridization. cDNA libraries, derived from a variety ofhuman and/or mouse tissues, and propagated in lambda phage vectors (eg.,lambda gt11) were plated on agarose, plaques were transferred to nylonmembranes and probed with fragments derived from the unique regions ofthe FKH^(sf) gene. Under high stringency conditions (eg., hybridizationin 5×SSPE, 5×Denhardt's solution, 0.5% SDS at 65° C., washed in0.1×SSPE, 0.1% SDS at 65 C) only very closely related sequences areexpected to hybridize (i.e., 90-100% homologous). Under lowerstringency, such as hybridization and washing at 45°-55° C. in the samebuffer as above, genes that are related to FKH^(sf) (65-90% homologous)may be identified. Based on results obtained from searching publicdatabases with the unique sequences of FKH^(sf) any genes identifiedthrough low- to mid-stringency hybridization experiments are expected torepresent novel members of a “FKH^(sf) family”.

Example 12 Overexpression of the Wild-Type Foxp3 Gene Results inDecreased Numbers of Peripheral T Cells

The original breeding stocks for scurfy mice were obtained from OakRidge National Laboratory (ORNL), with mice subsequently derived bycaesarian section into SPF conditions. Transgenic mice were generated byoocyte microinjection by DNX Transgenic Services (Cranbury, N.J.), asdescribed (Brunkow et al., Nat. Gen. 27:68-72, 2001). For the 2826 mouseline, a 30.8 kb cosmid construct was generated from mouse BAC K60 forinjection. This cosmid contains the entire Foxp3 gene along withapproximately 18 kbp of 5′ sequence and 4 kbp of 3′ sequence. Expressionof the gene parallels that of the endogenous gene with respect to tissuedistribution (Brunkow et al., Nat. Gen. 27:68-72, 2001). The Ick-Foxp3transgenic animals were generated using the Ick pacmotor to driveexpression (Garvin et al., Int. Immunol 2(2):173, 1990). Both transgenicand scurfy mice were backcrossed onto the C57B1/6 background (JAX) for4-6 generations for all studies. No differences in responsiveness orphenotype were noted during backcrossing. Northern blot analysis wasperformed as described previously (Brunkow et al., Nat. Gen. 27:68-72,2001).

Initial experiments involving the Foxp3 transgenic mice demonstratedthat in 5/5 lines generated from distinct founder animals, theexpression of the wild-type Foxp3 transgene prevented disease in sf/Ymutant mice (Brunkow et al., Nat. Gen. 27:68-72, 2001). Further analysisdemonstrates that the copy number of the transgene is directlycorrelated to the expression of the gene at the mRNA level (Brunkow etal., Nat. Gen. 27:68-72, 2001). This is likely due to the fact that thetransgene construct consisted of a large genomic fragment including asubstantial portion of 5′ sequence and much of the regulatory region. Inanalyzing the various transgenic lines, it also becomes clear that therewas a direct relationship between the expression of the Foxp3 gene andthe number of lymph node cells (Brunkow et al., Nat. Gen. 27:68-72,2001). The relationship between transgene copy number and cell number isshown for three of the founder lines, with the scurfy mutant animal(sf/Y) and normal littermate controls (NLC) for comparison (see, Table 1below). Lymphoid cell number from transgenic (lines 2826, 1292 and2828), normal littermate control and scurfy mutant (sf/Y) mice weredetermined for various tissues from representative age-matched (4 weekold) mice. The approximate transgene copy number was determined bySouthern blot analysis and correlated well with Foxp3 gene expression(Brunkow et al., Nat. Gen. 27:68-72, 2001). Although there is a lessdramatic, but consistent, difference in the number of splenic cells inthe transgenic mice as well, the number of thymocytes is notsignificantly affected. For reasons of simplicity, except where noted,the remainder of the experiments utilized the 2826 transgenic line.Animals from this line are generally healthy and survive for greaterthan one year under SPF conditions. The line has approximately 16 copiesof the transgene and by northern blot analysis is expressed at ten totwenty times the level of the endogenous gene in lymphoid tissues(Brunkow et al., Nat. Gen. 27:68-72, 2001). The transgene, like theendogenous gene, is only poorly expressed in non-lymphoid tissues, alikely consequence of its expression under the control of its endogenouspromoter. Lymph node cell number in mice from this line range from 15-50percent of normal, with the number of cells accumulating with age.Splenic cell number is less dramatically affected although generallydecreased, with a range of 25-90 percent of normal.

TABLE 1 Transgene Copy Cell Number (×10⁶) Genotype Number Thymus LymphNode Spleen NLC NA 121.4 1.5 84.4 2826 ~16 111.8 0.5 60.8 1292 ~9 98.61.0 76.4 2828 ~45 108.5 0.4 61.1 Scurfy NA 64.4 4.7 109.5

Example 13 Thymic Phenotype of Scurfin-Transgenic Mice

The role of the Foxp3 gene in thymic selection remains unclear. Deletionof superantigen-specific Vβ-bearing thymocytes appears normal in bothsf/Y as well as 2826 transgenic mice. Consistent with this,overexpression of the Foxp3 gene using its own endogenous promoter (2826line) also does not appear to result in any gross changes in thymicdevelopment or selection. The number of thymocytes (Table I) and theirdistribution amongst the major phenotypic subsets is indistinguishablefrom littermate control animals. Thymus, lymph node and splenic tissueswere collected as described (Clark et al., Immunol 162:2546, 1999) andwere resuspended in staining buffer (1% BSA, 0.1% sodium azide in PBS)at a cell density of 20×10⁶/mL. Cell aliquots were treated with 2%normal mouse serum (Sigma) to block non-specific binding then stained byincubation on ice for 30 minutes with combinations of the followingfluorochrome-conjugated anti-mouse monoclonal antibodies (mAbs): CD3,CD8β, CD4, CD25, IgG2a control (Caltag Laboratories, Burlingame,Calif.); CD28, CD45RB, CD44, CD62L, CD69, CD95 (PharMingen, San Diego,Calif.). The fluorescence intensity of approximately 10⁵ cells wasexamined using a MoFlo™ flow cytometer (Cytomation, Fort Collins, Colo.)with dead cell exclusion by addition of propidium iodide (10 μg/mL).

A more detailed examination of the CD4⁻8⁻ subset also reveals a normaldistribution of gamma-delta cells and CD25⁺ cells. Importantly, thefraction of CD4⁺8⁻ thymocytes expressing the maturation markers CD69 andHSA is identical in 2826 and control animals, suggesting that thematuration process is normal.

Overexpression of the Foxp3 gene in the thymus alone has a significantlydifferent phenotype from the 2826 mice noted above. Transgenic miceexpressing Foxp3 selectively in the thymus (16.5 and 8.3) under controlof the Ick proximal promoter were crossed to sf/+ carrier females. Malescurfy mice (sf/Y) that carried the thymus-specific transgene (16.5 and8.3) succumbed to disease at the same time and in the same manner asnon-transgenic littermates. Sf/Y transgenic animals expressing Foxp3under its endogenous regulatory sequences (2826) did succumb to disease.Cell number is derived from mice that carried the transgene in additionto the wild-type Foxp3 gene.

Transgenic animals that express the Foxp3 gene exclusively in thymus(under the control of the Ick proximal promoter) are unable to rescuesf/Y mice from disease (see, Table 2 below). Two separate founderanimals were crossed to scurfy carrier females in an attempt to preventdisease. In each case sf/Y mice carrying the Ick proximal promoter—Foxp3transgene developed an acute lymphoproliferative disease that wasidentical both in severity and time course to that seen innon-transgenic sf/Y siblings. In each case expression of the transgenewas restricted to the thymus with no detectable expression in peripheralorgans, including spleen. The Northern Blot analysis was carried out aspresented in Example 1. Further, thymic expression of the Ick-driventransgene was substantially greater than that of the gene in 2826transgenic animals or of the endogenous gene in normal littermatecontrol mice. Hence it appears that the fatal lymphoproliferativedisease seen in sf/Y mice does not arise as a consequence of scurfinmediated developmental defects in the thymus.

TABLE 2 Disease in Cell Number (×10⁶) Genotype Sf/Y mice? Thymus LymphNode NLC NA 79.0 2.9 2826 No 100.1 2.2 16.5 Yes 110.4 2.7 8.3 Yes 32.22.9

Although transgenic (non-sf) animals carrying the Ick-driven transgeneappear generally normal, high level expression of the transgene withinthe thymus does have phenotypic consequence in normal (non-sf) animals.Significantly increased expression of the transgene in otherwise normalmice leads to a relative decrease in the percentage of double-positivethymocytes and a corresponding increase in the percentage ofdouble-negative (DN) cells, as well as a decrease in overall thymic cellnumber (see, Table 2). T cell development still occurs in these animalsas assessed by the generation of CD4 and CD8 single positive cells andby the presence of relatively normal numbers of peripheral T cells inboth lymph node and spleen (see, Table 2). CD69 expression on CD4⁺8⁻cells from the thymus is similar in transgenic and wild-typelittermates, suggesting positive selection likely proceeds normally,whereas within the DN compartment, the fraction of cells expressing CD25is diminished relative to wild-type animals. These transgenic animalsindicate that overexpression of the Foxp3 gene within the thymiccompartment specifically can alter thymic development, but this appearsto have no effect on regulating peripheral T cell activity.

Example 14 Altered Phenotype of Peripheral T Cells fromScurfin-Transgenic Mice

In addition to a decrease in the number of peripheral T cells in 2826mice, there is a slight reduction in the percentage of CD4⁺8⁻ cells inboth the lymph node and spleen relative to NLC. Whereas the CD3 levelsappear normal on peripheral T cells, there are a number of other surfacemarkers with altered expression levels. For CD4⁺8⁻ cells in thetransgenic mice, the most consistent changes are a small decrease in theexpression of CD62L and CD45RB as well as an increase in the expressionof CD95. By comparison, cells from sf mutant animals have a verydifferent phenotype. CD4⁺8⁻ cells from these mice are large and clearlyactivated. They are predominantly CD44^(H1), CD45RB^(LO), CD62L^(LO) andpartially CD69⁺ (Clark et al., Immunol 162:2546).

CD4⁻8⁺ cell numbers were also reduced in both the spleen and lymph nodesof scurfin-transgenic mice. This decrease is typically more dramatic!(50-75%) than the decrease in the CD4⁺8⁻ compartment (25-50%). CD4⁻8⁺ Tcells display relatively minor and variable changes in the level ofCD62L, CD45RB and CD95 on the cell surface in comparison to NLC. Incontrast to CD4⁺8⁻ T cells, there is a more pronounced increase in thepercentage of CD4⁻8⁺ T cells that were also CD44^(H1). Overall, theCD4⁻S⁺ cells do not express surface markers at levels that characterizethem as specifically naive, activated or memory.

Example 15 Histological Analyses of Scurfin-Transgenic Mice

Whereas peripheral T cells in 2826 mice are clearly decreased in number,a determination was made whether the architecture of the lymphoid organswas also perturbed. Histological examination of the major lymphoidorgans (thymus, lymph node and spleen) indicated that the mostsignificant changes were found in the mesenteric and peripheral lymphnodes. Tissues for histological analysis were removed from miceapproximately 8 weeks after birth and immediately fixed in buffered 10%formalin. Paraffin-embedded sections were processed for hematoxylin andeosin staining and comparative histopathology performed onrepresentative mice. As expected, the thymus appears relatively normal,with a well-defined cortico-medullary junction, although there appearsto be a slight reduction in the size of the thymic medulla. Transgenicanimals had smaller peripheral lymph nodes, lack robust and normallydistributed lympoid follicles, lack distinct margins between follicularand interfollicular areas and had more obvious sinuses than those foundin the lymph nodes of the normal littermate control mice. Even thoughthe spleen and Peyer's patches appear approximately normal in size andmicroarchitecture, there is a moderate decrease in total cell number andno or minimal evidence of germinal centers in these tissues. The changesnoted here reflect a hypocellular state distinct from a number of othertargeted mutations in which the lymph nodes fail to develop. Therefore,while T cells are capable of development in an apparently normal manner,their representation within the peripheral lymphoid tissues,particularly the lymph nodes, is substantially decreased.

Example 16 Decreased Functional Responses of CD4⁺8⁻ Cells fromScurfin-Transgenic Mice

The phenotypic and cell number data suggest that there are specificdefects in the biology of CD4 T cells from 2826 transgenic animals. Thefunctional responses of T cells from these animals to several stimuliwere evaluated, including anti-CD3 and anti-CD28. Lymphocytes wereisolated from various tissues from NLC, 2826 transgenic or scurfy(mutant) mice and CD4 cells were purified by cell sorting. Thymus, lymphnode and splenic tissues were removed from appropriate animals,macerated between sterile microscope slides, filtered through a sterile70 μm nylon mesh and collected by centrifugation. CD4⁺ T lymphocyteswere sort purified from these tissues by positive selection using theMoFlo. Sort purities as determined by post-sort analysis were typicallygreater than 95%. Cells were cultured at 37° C. in complete RPMI (cRPMI)(10% fetal bovine serum, 0.05 mM 2-mercaptoethanol, 15 mM HEPES, 100U/mL penicillin, 100 μg/mL each streptomycin and glutamine) in 96-wellround-bottomed tissue culture plates. Culture wells were prepared for Tcell activation by pre-incubation with the indicated concentrations ofpurified antibody to CD3 (clone 2C11) in sterile PBS for 4 hours at 37°C. Purified α-mouse CD28 (clone 37.51) or α-mouse KLH (control antibody)was co-immobilized at 1 μg/ml final concentration.

T cells were cultured at a cell density of 1 to 5×10⁴ cells/well in 200μL of cRPMI for 72 hours. Supernatant (100 μl) was removed at 48 hoursfor analysis of cytokine production. Wells were pulsed with 1 μCi/wellof ³[H]-thymidine (Amersham Life Science, Arlington Heights, Ill.) forthe last 8-12 hours of culture and then harvested (Tomtec).Proliferation data reported are based upon mean value of triplicatewells and represent a minimum of 3 experiments. Cytokine levels weredetermined by ELISA assay according to the manufacturer's direction(Biosource International, Camarillo, Calif.).

To test for proliferation and IL-2 production, a single cell suspensionof Balb/c spleen cells was generated to used as stimulator cells. Thesecells were irradiated (3300 rads) and incubated a 10:1 ratio(stimulator:effector) with scurfin-transgenic or NLC spleen cells. Tosome cultures, IL-2 was added at 100 U/ml. For proliferation assays,cells were pulsed after five days and harvested as above. Bothproliferation and IL-2 production are significantly diminished in cellsfrom the transgenic animals compared to their littermates. Althoughtransgenic animals increase their responsiveness with increasingstimulation, they rarely reached the levels achieved by NLC. This isparticularly true for IL-2 production, in which cells from 2826 miceconsistently produce low to undetectable amounts of this cytokine.Similar results were seen whether the cells were derived from the spleenor the lymph nodes.

As expected, cells from scurfy animals were hyper-responsive tostimulation and produce increased amounts of IL-2. The effect of thetransgene was independent of strain and have remained constant duringthe back-crossing of the animals onto C57BI/6 through at leastgeneration N6. T cells from transgenic mice remained responsive toanti-CD28 in this assay whereas stimulation with anti-CD3 and control Igresults in generally poor responses that were lower than, but similar toNLC responses. Addition of high doses of IL-2 is able to partiallyovercome the proliferative defect in CD4⁺8⁻ T cells from 2826 mice, butgenerally fails to restore the response to that of wild-type animals.

In contrast to peripheral T cells, but consistent with the phenotypicdata above, the proliferative response of thymic CD4⁺8⁻ cells isapproximately comparable between transgenic and NLC mice. IL-2production by thymic CD4⁺8⁻ cells however, is reduced substantially fromthe transgenic animals. The reduction in IL-2 production by thymocytesis somewhat more variable than that seen in lymph node or spleen and maysuggest that the IL-2 produced is also consumed during the culture.Alternatively, thymocytes may produce other growth factors less affectedby the expression of the Foxp3 gene. Nevertheless, the data generallysupport the conclusion that a major defect in the transgenic animals isin the ability of both thymic and peripheral T cells to produce IL-2.

Example 17 Altered Functional Responses of Scurfin-Transgenic CD4⁻8⁺ TCells

The ability of transgenic T cells to generate and function as cytotoxicT cells (CTL) was determined in an in vitro assay. A single cellsuspension of Balb/c spleen cells was generated to use as stimulatorcells. These cells were irradiated (3300 rads) and incubated a 10:1ratio (stimulator:effector) with scurfin-transgenic or NLC spleen cells.To some cultures, IL-2 was added at 100 U/ml. For generation of CTL,splenic T cells were stimulated in a similar manner in the presence of100 U/ml of IL-2. After five days, cells were either assayed in the JAMassay (Matzinger, P. J Immunol 145(1-2):185 (1991)) or re-stimulated ona new stimulator layer. Cells were approximately 95% CD4⁻8⁺.

Transgenic T cells were stimulated in a mixed-lymphocyte culturecontaining increasing numbers of irradiated allogeneic stimulator cellsin the presence or absence of IL-2. The proliferative response of eithertransgenic or NLC effector cells was then measured. T cells from thetransgenic animals responded poorly in the absence of exogenous IL-2,consistent with the data for purified CD4⁺8⁻ cells (above). In thepresence of exogenous IL-2, transgenic T cells displayed an increasedproliferative response, but still required a higher number of stimulatorcells to reach a similar level of proliferation as control cells. Theability of mixed T cell populations to respond to stimulation in thisassay may reflect the presence of both CD4⁺8⁻ and CD4⁻8⁺ T cells inthese cultures.

As a direct indicator of CD4⁻8⁺ activity, the cytotoxic ability of Tcells were assayed in a standard target cell lysis assay. CD4⁻8⁺ T cellswere generated using allogeneic feeder cells in the presence of IL-2 andassayed to determine the ability of these cells to lyse target cells.Balb/c spleen cells were stimulated with PMA (10 ng/ml) in the presenceof ionomycin (250 ng/ml) for 24 hours to allow for efficient loading ofcells with ³[H]-thymidine. After 24 hours, ³[H]-thymidine (5 μCi/ml) wasadded to PMA+Ionomycin-stimulated Balb/c spleen cells. Cells wereincubated at 37° C. for 18 hours and then washed. CD4⁻8⁺ effector cellswere plated with target Balb/c cells at increasing ratios ranging from1.5:1 to 50:1 (effector:target) in a 96-well flat-bottom plate(experimental) in a final volume of 100 μl. The cells were pelleted bycentrifugation and incubated at 37° C. for four hours. A platecontaining labeled Balb/c cells alone was harvested immediately and usedto determine total counts (TC). A second plate containing labeled Balb/ccells alone was also incubated at 37° C. for four hours to determinespontaneous release (SR). After four hours of incubation, cells wereharvested onto glass fiber and counted in a scintillation counter.

Percent lysis was determined as follows:{[(Total-SR)−(Experimental−SR)]/(Total counts−SR)}*100=% lysis. Athigher effector-to-target ratios (50:1 and 25:1), scurfin-transgenicCD4⁻8⁺ cells were as effective at lysing target cells as cells generatedfrom NLC, while at the intermediate ratios (12.5-3:1), transgenic cellswere significantly reduced in their cytolytic function in comparison toNLC. However, the transgenic cells were still effective with 50-60%lysis at these intermediate ratios. Overall, these data suggest thatscurfin-transgenic T cells possess cytolytic activity, but are lesseffective than NLC. In addition, exogenous IL-2 was required to generatefunctional CD4⁻8⁺ T cells, presumably due to the poor endogenousproduction of this cytokine.

As a further indicator of T cell responsiveness, the functionalresponsiveness of 2826 transgenic animals to antigen in vivo wasaddressed. Contact sensitivity responses using Oxazalone as thechallenging agent were carried out on 2826 mice and their littermatecontrols. Age-matched animals were treated on the left ear with 2%Oxazalone (diluted in olive oil/acetone), using a final volume of 25 μl.After 7 days, ear thickness was measured using spring-loaded calipersand mice were challenged on the right ear with 2% Oxazalone (8 μl perear). Ear thickness was measured at 24 hours and is reported as changein ear thickness compared to pre-challenge. Control mice were challengedonly. Thickness of ears following initial priming (prior to challenge)was no different from untreated ears. Mice were subsequently treatedwith PMA (10 ng/ml; 8 μl/ear) on the priming ear. Ear thickness wasmeasured at 18 hours and is reported as thickness compared topre-treatment.

In these studies, transgenic animals made a consistently poor responseto Oxazalone at all times examined, whereas control animals respondednormally. The transgenic animals however responded normally to challengewith PMA, indicating that they were capable of generating aninflammatory reaction to a strong, non antigen-specific challenge.Further studies using animals transgenic for both a TCR and Foxp3 willexamine in vivo responses in greater detail.

Example 18 Scurfy T Cells can be Inhibited by Wildtype T Cells In Vivo

It has previously been reported that adoptive transfer of CD4⁺8⁻ T cellsfrom sf mice into nude mice transfers disease as measured by the wastingand skin lesions characteristics of sf. However, grafting of sf thymusinto normal mice does not transmit the disease suggestingimmunocompetent mice are capable of inhibiting sf cells (Godfrey et al.,Am. J. Pathol. 145:281-286, 1994). To better understand the mechanism ofinhibition either 3×10⁶ sf CD4+ T cells or wildtype CD4⁺ T cells or amix of sf and wildtype CD4⁺ T cells were adoptively transferred intosyngeneic C3H-SCID mice.

C3H SCID mice were purchased from The Jackson Laboratory (Bar Harbor,Me.). All animals were housed in specific pathogen free environment andstudies were conducted following PHS guidelines. The original doublemutant strain, sf (sf) and closely linked sparse-fur (Otc^(spf)), wereobtained from Oak Ridge National Laboratory. The double mutants werebackcrossed to Mus musculus castaneous to obtain recombinants carryingeither (Otc^(spf)) mutation or sf mutation (Brunkow et al., Nat Gen27:68-72, 2001). Prior to cloning of sf gene carrier females for sfmutation were identified by the amplification of genomic DNA withprimers 5′-ATTTTGATT ACAGCATGTCCCC-3′ (SEQ ID NO:15) and5′-ACGGAAACACTCTTATGTGCG-3′ (SEQ ID NO:16) (primers for microsatellitemarker DXMit136 which was found to be inseparable from sf phenotypeduring backcrossing).

The single mutant sf strain was maintained by breeding carrier femalesto F1 males of (C3Hf/rI×101/RI) or (101/RI×C3H/RI). Sf males were usedat age 15-21 days and wildtype control animals were used at 6-12 weeksof age. Scurfy or wildtype CD4⁺ T were purified by cell sorting. Thecells were resuspended in 0.9% saline, pH 7.2 and mixed at differentratios in a final volume of 200 μl and injected into SCID mice viatail-vein. Mice were monitored weekly for weight loss. Approximately 50μl of blood was collected by eye-bleeds. Red blood cells were lysed andleukocytes were stimulated at 5×10⁴ cells/well for 48 hours withimmobilized anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml).

Mice that received sf T cells showed signs of wasting (seen as weightloss) 3-4 weeks post-transfer that became progressively worse, whereasthe mice that received a mixture of sf and wildtype T cells showed anormal weight gain corresponding to their age (FIG. 11A). Mice thatreceived only wildtype T cells showed a similar weight gain with age. Inaddition, mice receiving only sf T cells developed an inflammatoryreaction around, but not within, the eye that persisted throughout theexperiment. If the disease was allowed to progress, the recipients of sfT cells only died 8-16 weeks after transfer. Recipients of a mix of sfand wildtype T cells remained healthy throughout the experiment(experiments done up to 16 weeks).

Histological examination of the large intestine of mice receiving sf Tcells showed crypt abscesses, thick epithelium, increased epithelialcellularity and cellular infiltrates in the colonic wall, consistentwith proliferative colitis (FIG. 11B). In comparison, the intestine ofmice receiving a mixture of sf and wildtype T cells (or wildtype cellsalone) appeared normal, correlating with the lack of wasting in thesemice.

For the histological examination, tissues were removed from C3H/SCIDmice receiving either sf T cells, wildtype T cells or a mixture of sfand wildtype T cells. Intestines were flushed with cold PBS andimmediately fixed in 10% formalin. Paraffin embedded sections wereprocessed for hematoxylin and eosin staining and comparativehistopathology (Applied Veterinary Pathobiology, Bainbridge Is., WA).

Cellular infiltration and inflammation was also noted in a number ofother organs (including kidney, liver and skin) from mice that receivedsf T cells only and such cells were not found in animals that receivedwildtype cells. Further, the lymph nodes and spleen from sf-recipientanimals were substantially enlarged compared to their controls,indicating a marked lymphoproliferative process. Lymph nodes werecollected from 6-12 weeks old mice and macerated in DMEM+10% FBS inbetween the ground glass ends of sterile microscope slides. The cellswere filtered through 70 μM nylon mesh, collected by centrifugation andresuspended at ˜50×10⁶ cells/ml in complete media.

CD4⁺ T cells from sf mice have been shown to be hyperproliferative andto secrete large amounts of cytokines such as IL-2, IL-4 and IFN-γ(Blair et al., J. Immunol. 153:3764-774 (1994); Kanangat et al., Eur. J.Immunol. 26:161-165 (1996)). To monitor the activation status of theCD4⁺ T cells that were transferred into the SCID animals, IL-4 secretionby PBMC of recipient mice was measured. PBMC from various recipientswere stimulated with anti-CD3 and anti-CD28 in vitro for 48 hours andsecreted IL-4 was detected by ELISA kit (BioSource International,Camarillo, Calif.) according to manufacturer's instruction. At an earlytime point post-transfer (8-10 days), IL-4 was produced by PBMC from allthe recipients (FIG. 11 c). At later time points (2 weeks or more), PBMCfrom recipients of sf T cells secreted significant amounts of IL-4whereas the PBMC of mice receiving either wildtype T cells only or amixture of sf and wildtype T cells secreted little IL-4. Lack of weightloss, tissue infiltrates and suppression of IL-4 secretion in micereceiving a mixture of sf and wildtype T cells indicated that wildtype Tcells were inhibiting the activation and disease progression normallyassociated with the transfer of sf CD4+ T cells.

Example 19 Sf Cells are Regulated by CD4⁺ CD25⁺ T-Regulatory Cells

There have been numerous reports that the CD4⁺CD25⁺ subset of peripheralCD4⁺ T cells (T-reg cells) is involved in regulating other T cells, bothin vivo and in vitro (Roncarolo et al., Curr. Opin. Immun. 12:676-683(2000); Sakaguchi, S., Cell 101:455-458 (2000); Shevach, E. M., Ann.Rev. Immun. 18:423-449 (2000)). It was therefore of interest todetermine if such T-reg cells were responsible for the inhibition ofdisease seen after co-transfer of sf and wildtype CD4⁺ T cells in vivo.Two million sf CD4⁺ T cells were mixed at different ratios either withwildtype CD4⁺CD25⁻ T cells or with wildtype CD4⁺CD25⁺ T-reg cells andinjected into C3H/SCID mice. The recipients were monitored for weightloss and IL-4 secretion by PBMC as described in Example 1. For isolatingT-reg cells these were stained with anti-CD4-FITC (Caltag Laboratories,Burlingame, Calif.) and anti-CD25-biotin (Caltag) for 30 min on ice. Thecells were washed twice with PBS and stained with strepavidin-APC(Molecular Probes, Eugene, Oreg.) for 20 min on ice. Cells were washedtwice and positive sorted for CD4⁺CD25⁺ T cells.

As before, mice receiving sf T cells alone showed signs of wasting (FIG.12) and IL-4 production. However, mice that received a mixture of sf Tcells and higher doses (110,000 or more) of wildtype CD4⁺CD25⁺ T-regcells showed a marked reduction in signs of disease such as weight loss.In comparison, mice that received a mix of sf and CD4⁺CD25⁻ T cellsshowed signs of disease at all doses except when the number of CD4⁺CD25⁻T cells was greater than 1.1 million. The small amount of suppressionseen with higher numbers of CD4⁺CD25⁻ T cells may indicate that thereare additional mechanisms of suppression or that CD4⁺CD25⁻ T cells giverise to CD4⁺CD25⁺ T-reg cells post-transfer. It seems unlikely that themechanism of inhibition by CD4⁺CD25⁻ T-reg involves in vivo competitionfor lymphoid space, since as few as 1.1×10⁵ T-reg can inhibit theactivity of 2×10⁶ sf T cells.

In order to better understand the mechanism of inhibition of sf CD4⁺ Tcells by CD4⁺CD25⁺ T-reg cells, in vitro mixing experiments wereconducted. Sf CD4⁺ T cells or wildtype CD4⁺CD25⁻ T cells were activatedwith anti-CD3 in presence or absence of CD4⁺CD25⁺ T-reg cells at variousresponder:suppressor ratios. FIG. 13 shows that the proliferativeresponses of wildtype CD4⁺ T cells stimulated with APC and anti-CD3 weresuppressed significantly by the addition of CD4⁺CD25⁺ T-reg cells. TheseCD4⁺CD25⁺ T-reg cells also inhibited the proliferative responses of sfCD4⁺ T cells. However CD4+CD25+ T-reg cells were less effective ininhibiting sf CD4⁺ T cells than wildtype CD4⁺ T cells. This result, likethat seen with co-transfer in vivo, indicates that the hyper-responsivestate of sf T cells can be regulated by T-reg cells.

For APC, spleens were collected in a similar fashion as lymph nodes andstained with anti-Thy-1-FITC or anti-Thy1-PE (Caltag). Cells were washedand negative sorted for Thy-1. The cells were sorted using a MoFlo flowcytometer (Cytomation, Fort Collins, Colo.) and Cyclops (Cytomation)software at a rate of 10-20,000/min. Cell doublets and monocytic cellswere eliminated on the basis of forward and side scatter gates, and deadcells were excluded by propidium iodide (10 μg/ml) stain. The purity ofthe sorted cell population was routinely 90-99%. Thy-1⁻ APC were treatedwith mitomycin C (Sigma, 50 μg/ml) for 20 min at 37° C. and washed threetimes with DMEM+10% FBS before using in proliferation assays. Forregulatory T cell assays, CD4+ T cells were stimulated at 5×10⁴cells/well in 200 ul DMEM+10% FBS with soluble anti-CD3 (2C11;Pharmingen) at 1 μg/ml and an equal number of mitomycin C treated Thy-1⁻APC from spleens. For T-reg assays MoFlo sorted CD4⁺CD25⁺ T cells wereadded at various ratios.

Cultures were incubated 72 hour at 37° C. and pulsed with 1 μCi/wellwith [³H] thymidine (Amersham Life Sciences, Arlington, Ill.) for thelast 8-12 hours of culture. For in vitro preactivation, CD4⁺CD25⁺ orCD4⁺CD25⁻ T cells were stimulated at 5×10⁴ cells/well in 200 μl DMEM+10%FBS with soluble anti-CD3 (1 μg/ml for wildtype cells or 10 μg/ml forFoxp3 transgenic cells), 4 ng/ml rIL-2 (Chiron) and an equal number ofmitomycin-C treated Thy-1⁻ APC from spleens. The cells were harvested at72 hours, stained with CD4-FITC or CD4-PE and positively sorted for CD4using a MoFlo flow cytometer as described above. These cells were thenadded to the regulatory T cell assay as previously described at the sameratios as freshly isolated CD4⁺CD25⁺ T cells.

Example 20 TGF-β does not Inhibit SF CD4⁺ T Cells

Recent studies have implicated a critical role for CTLA-4 and secretionof TGF-β in regulatory function of CD4⁺CD25⁺ T-reg cells in vivo (Readet al., J. Exp. Med. 192:295-302, 2000); Takahashi et al., J. Exp. Med.192:303-310, 2000). To test whether sf cells were sensitive toinhibition with TGF-β, CD4⁺ T cells were stimulated with or without theaddition of exogenous TGF-β. For the TGF-β assays, anti-CD3 (at varyingconcentrations, Pharmingen) and anti-CD28 (1 μg/ml, Pharmingen) wereimmobilized on plastic. TGF-β (R&D) was added at a final concentrationof 2.5 ng/ml. Cultures were incubated for indicated time periods at 37°C. Individual wells were pulsed with 1 μCi/well with [³H] thymidine(Amersham Life Sciences, Arlington, Ill.) for the last 8-12 hours ofculture. Proliferation data are mean value of triplicate wells andrepresent a minimum of three experiments.

As expected, wildtype CD4⁺ T cells stimulated with either anti-CD3 alone(FIG. 14A) or with the combination of anti-CD3 and anti-CD28 wereinhibited significantly by TGF-β (FIG. 14B). However, sf cellsstimulated either with anti-CD3 or anti-CD3/CD28 were not sensitive toinhibition with TGF-β, regardless of the dose of anti-CD3 or TGF-β. Thelack of TGF-β, inhibition was specific to T cells since proliferationand cytokine production by B cells as well as monocytes, both stimulatedwith LPS, from sf mice were sensitive to TGF-β inhibition. It is worthnoting however, that high levels of exogenous IL-2 can largely overcomethe inhibitory effect of TGF-β on T cells, potentially by downregulatingTGF-β receptor expression (Cottrez et al., J. Immunol. 167:773-778,2001). T cells from sf animals produce extremely high levels of IL-2upon stimulation, and this may contribute to the lack of inhibition byTGF-β on T cell function of sf mice. Additionally, the role of TGF-βproduction by T-reg cells in in vitro assays is not clear. Mostexperimental systems do not point to a role for TGF-β in this system,although the in vivo data does indicate an important role for TGF-β ininhibitory activity of CD4⁺CD25⁺ T-reg cells (Read et al., J. Exp. Med.192:295-302, 2000).

Example 21 Foxp3 Expression is Upregulated in CD4⁺CD25⁺ T-Reg Cells

The Foxp3 gene is expressed at highest levels in lymphoid tissues suchas thymus, lymph node and spleen (Brunkow et al., Nature Genetics27:68-72, 2001). The lymphoid expression of Foxp3 seems to bepredominantly in CD4⁺ T cells, since the level of expression in CD8⁺ Tcells as well as B cells was significantly lower or undetectable(Brunkow et al., Nature Genetics 27:68-72, 2001).

To assess if Foxp3 plays a role in CD4⁺CD25⁺ T-reg cells, the expressionof Foxp3 transcript in CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells from normal aswell as Foxp3 transgenic mice (˜16 copies of Foxp3 transgene) wascompared. CD4⁺CD25⁺ or CD4⁺CD25⁻ T cell populations were collected asdescribed above. Oligo dT primed first-strand cDNA was synthesized fromthese cells using the SuperScript Preamplification System (Gibco-BRL,Rockville, Md.) and used as a template for real-time RT-PCR using an ABIPrism 7700 instrument. Foxp3 expression was measured using the primers5′-GGCCCTTCTCCAGGACAGA-3′ (SEQ ID NO:17) and 5′-GCTGATCATGGCTGGGTTGT-3′(SEQ ID NO:18) at a final concentration of 300 nM and with internalTaqMan probe 5′-FAM-AGCTTCATCCTAGCGGTTTGCCTGAG-AATAC-TAMRA-3′ (SEQ IDNO:19) at a final concentration of 100 nM. Dad1 was used as anendogenous reference (Hong et al., 1997). Dad1 primers were5′-CCTCTCTG-GCTTCATCTCTTGTGT-3′ (SEQ ID NO:20) and5′-CCGGAGAGATGCCTTGGAA-3′ (SEQ ID NO:21), used at a final concentrationof 50 nM and TaqMan probe5′-6FAM-AGCTTCATCCTAGCGGTTTGCCTGAGAATAC-TAMARA-3′ (SEQ ID NO:22) at afinal concentration of 100 nM. Other components of the PCR mix were fromthe TaqMan Universal Master Mix (PE Applied Biosystems). PCR cyclingconditions were 50° C. for 2 min; 95° C. for 10 min; and 40 cycles of95° C. for 15 seconds, 60° C. for 1 minute.

Data was collected by ABI Prism 7700 Sequence Detection System Software,Version 1.6.4. A standard curve was generated with a dilution series(1×, 1:10, 1:100, 1:1000, 1:10,000) of a standard cDNA sample which wasrun at the same time as the unknown samples. The software determines therelative quantity of each unknown based by plotting a curve of thresholdcycle (C_(T)) versus starting quantity and using the C_(T) to calculatethe relative level of unknown sample. Each sample was run in duplicateand mean values used for calculations. Data is expressed as normalizedFoxp3 expression, which was obtained by dividing the relative quantityof Foxp3 for each sample by the relative quantity of Dad1 for the samesample.

Interestingly, the level of Foxp3 expression in CD4⁺CD25⁻ T cells wasnearly undetectable whereas CD4⁺CD25⁺ T-reg cells expressed the highestamounts of Foxp3 so far described (FIG. 15A). The level of Foxp3expression in T cell subsets of Foxp3 transgenic mice was alsodetermined. These animals have ˜16 fold the amount of Foxp3 messagefound in wildtype animals. In Foxp3 transgenic mice, Foxp3 expressionwas detectable in both CD4⁺CD25⁻ as well as CD4⁺CD25⁺ T cells, butsimilar to wildtype cells, CD4⁺CD25⁺ T cells expressed significantlygreater levels of Foxp3. A subset of CD4⁺ cells in sf mutant animalsalso expresses CD25, although this population is large in size andexpresses CD69, indicating they are likely cells previously activated invivo. Nonetheless, it was determined that these CD4⁺CD25⁺ cells from thesf mutant animals do not show enhanced amounts of Foxp3 message,indicating that these cells are likely not T-reg in nature.

CD4⁺CD25⁺ T-reg cells express certain markers such as CTLA-4, OX-40,GITR (McHugh, R. S. et al. Immunity 16: 311-23, 2002); Shimizu, J. etal. Nature Immunology 3: 135-42, 2002) that are characteristics ofactivated T cells. To assess if Foxp3 expression in CD4⁺CD25⁺ T-regcells was due to activation of T cells, Foxp3 expression was measured inCD25+ and CD25− subsets of CD4 T cells before and after in vitroactivation (FIG. 15B). CD4⁺CD25− T cells did not express any Foxp3 evenafter in vitro with anti-CD3 and IL-2. Interestingly, the expression ofFoxp3 in CD4⁺CD25⁺ T-reg cells decreased slightly after activation. Thisindicated that Foxp3 unlike any other markers reported so far onCD4⁺CD25⁺ T-reg cells was specific to this subset and was unrelated tothe activated/memory phenotype of these cells.

Example 22 Overexpression of Foxp3 Leads to an Increased Number ofCD4⁺CD25⁺ Cells But does not Lead to an Increase in Regulatory Activity

The relatively exclusive expression of Foxp3 within the T-reg subsetmight indicate that this transcription factor is either required for thegeneration of this subset or is directly involved in its function. Todetermine if Foxp3 plays a role in CD4⁺CD25⁺ T-reg cell function, thefunctional activity of CD4⁺CD25⁺ and CD4⁺CD25⁻ T cell subset from Foxp3transgenic mice was examined. These animals have 16 fold more Foxp3message than found in wildtype animals, with very high amounts in theCD4⁺CD25⁺ subset. Additionally, there were fewer total CD4⁺ cells inthese transgenic animals and those cells are hyporesponsive relative totheir littermate controls. Whereas there were a slightly increasedpercentage of CD4+CD25+ T cells in the transgenic mice, the expressionof CD25 was more diffuse and, unlike in wildtype animals, these cellsdid not comprise a distinct subset of cells (FIG. 16). A comparison offunctional activity of CD4⁺CD25⁺ T-reg cells from wildtype and Foxp3transgenic mice showed that although cells from the transgenic mice dodisplay regulatory activity, there was no significant increase insuppressive ability relative to their wildtype counterparts on aper-cell basis (FIG. 17).

Under the T-reg assay conditions the CD4⁺CD25⁺ T-reg cells wereactivated at the same time as the responders. Since CD4⁺ T cells fromFoxp3 transgenics were hyporesponsive to TCR stimulation it was likelythat the Foxp3-Tg CD4+CD25+ T-reg cells were not getting activated tothe same extent as the wildytpe CD4⁺CD25⁺ T-reg cells during the assay.This raised the possibility that if CD4⁺CD25⁺ T-reg cells from Foxp3transgenics were activated to the same extent as wildtype cells theywould exhibit higher regulatory activity.

To address the issue CD4⁺CD25⁺ T cells were pre-activated in vitro withanti-CD3 in the presence of APC and IL-2 for 72 hours according to thepreviously published protocol (Thornton et al., J. Immun. 164:183-190,2000). Based on our previous observations the T cells from Foxp3transgenic mice were activated with a higher dose of anti-CD3 in vitroto give comparable proliferation as the wildtype cells. Thesepreactivated T cells were then tested in a T-reg assay. As reported byothers, preactivation of CD4⁺CD25⁺ T cells in vitro made them much morepotent suppressors. However, preactivation of Foxp3 transgenic T-regcells gave them comparable suppressor activity as wildtype T-reg cells(FIG. 17). This suggested that there was no intrinsic defect in T-regcells from Foxp3 transgenics however overexpression of Foxp3 beyond athreshold level did not further enhance T-reg activity.

Example 23 CD4⁺CD25⁻ T Cells from Foxp3 Transgenic Mice Show RegulatoryActivity

Since CD4⁺CD25⁻ T cells from Foxp3 transgenics express Foxp3 at levelshigher than wild-type CD4⁺CD25⁺ T cells, we next evaluated expression ofsurface markers associated with T regulatory cells and the suppressiveactivity of these cells. Interestingly, the CD4⁺CD25⁻ T cells from Foxp3transgenics also expressed GITR (TNFRSF18) that has recently been shownto modulate T-reg activity (FIG. 18). These cells did not express otheractivation associated T cell markers such as OX40, CTLA4 or Ly-6A/E(data not shown). More importantly, when freshly isolated CD4⁺CD25⁻ Tcells from Foxp3 transgenic were tested for function in T-reg assay theyhad significant suppressive activity (FIG. 19). This activity usuallyranged from comparable to lower than that of CD4⁺CD25⁺ T cells from thesame mice. As expected, such suppressive activity was never detectedwith wild-type CD4⁺CD25⁻ T cells. In contrast to the CD4⁺CD25⁺ T cells,the suppressive activity of CD4⁺CD25⁻ T cells from Foxp3 transgeniccould not be enhanced by preactivation with anti-CD3 and IL-2 in vitro(data not shown). This further supports the idea that the expression ofFoxp3 commits a T-cell to the T-reg lineage without a direct correlationwith regulatory activity.

The gene mutated in sf mice (Foxp3) has a critical role in theregulation of peripheral T cell responses. Loss of function mutations inthe gene leads to a potentially fatal T cell mediated autoimmune diseaseboth in mice and humans (Bennett et al., Nature Genetics 27:20-21(2001); Lyon et al., Proc. Nat'l. Acad. Sci. USA 87:2433-2437 (1990);Wildin et al., Nature Genetics 27:18-20 (2001)). Additionally,overexpression of scurfin in transgenic mice leads to decreasedperipheral T cell numbers and inhibition of a variety of T cellresponses including proliferation and IL-2 production. Inhibition ofIL-2 production by scurfin is not the sole explanation forhyporesponsivess since addition of exogenous IL-2 does not completelyrestore normal T cell response in mice overexpressing scurfin. To betterunderstand the immunoregulatory mechanisms that may be controlled by theFoxp3 gene, further studies were conducted on the expression of thisgene and the biological role of scurfin-expressing cells.

As shown in this Example, wildtype T cells can inhibit disease caused byadoptive transfer of sf CD4+ T cells into SCID mice. These observationsare very similar to those made by several other groups characterizingthe activity of regulatory T cells. Similar to the observation made byPowrie et al., the disease caused by sf cells was inhibited by even asmall number of CD4⁺CD25⁺ T cells. CD4⁺CD25⁻ T cells were less effectiveat inhibiting sf T cell activity in this model, which may be due to asubset of these cells developing into a T-reg cell subset and making theappropriate inhibitory factors or due to additional mechanisms ofinhibition. In addition, the in vitro hyper-responsive state of sf Tcells can be inhibited by the presence of wildtype CD4⁺CD25⁺ cells, butnot by the addition of TGF-β. Generally, data from in vitro T-regexperiments suggest a direct cell-cell interaction is required with noinvolvement of cytokines such as TGF-β (Thornton et al., J. Exp. Med.188:287-296 (1998); Thornton et al., J. Immun. 164:183-190 (2000)).Additionally, TGF-β has no inhibitory effect on activated T cells(Cottrez et al., J. Immunol. 167:773-778 (2001)) making it unlikely thatCD4⁺CD25⁺ T-reg cell inhibition of sf cells in vivo is mediated byTGF-β.

To assess whether the Foxp3 gene product plays a role in CD4+CD25+ T-regcell function we measured the expression of Foxp3 in CD4+CD25+ T-reg andCD4+CD25− T cells and measured the regulatory activity of CD4+CD25+T-reg cells from mice overexpressing the Foxp3 gene. In both wildtypeand Foxp3 transgenics, CD4+CD25+ T-reg cells expressed the highest levelof Foxp3 mRNA of all different cell populations tested to date. Acomparison of functional activity of CD4+CD25+ T-reg cells from wildtypeand Foxp3 transgenic mice showed no increase in regulatory activity incells from transgenic mice, even following an optimal stimulation ofthese cells. Importantly however, CD4+CD25− T cells from Foxp3transgenic animals did have suppressive activity. While it is notpossible to phenotypically identify a subset of T-reg cells in sf mutantmice (due to the high level of endogenous activation), CD4+CD25+ cellsisolated from mutant animals neither expressed the Foxp3 gene nor didthey display any suppressive activity in vitro.

These results indicate that although expression of Foxp3 can commit a Tcell to the T-reg cell lineage, over expression of Foxp3 beyond athreshold level does not lead to further enhancement of regulatoryactivity. Furthermore, expression of Foxp3 by itself is likely notsufficient to generate T-reg cells, as CD4+CD25− from Foxp3 transgenicmice have comparable Foxp3 expression to wild-type T-reg cells but lesssuppressive activity. This effect on regulatory activity is unlikely dueto an effect on CTLA-4 expression since there is no increase in CTLA-4expression in Foxp3 transgenic mice and sf mutant animals express normallevels of CTLA-4.

Example 24 Modulation of Scurfin Expression

Antibodies or NCEs that modulate scurfin expression are identified usingthe following methods:

The scurfin promoter is cloned into commercially available Luciferasereporter vector (Promega, Madison, Wis.). This construct is thentransfected into cells, such as a murine or human T cell line. Agents,such as antibodies generated against T cells, cytokines, receptors, orother proteins, in addition to small molecules, peptides, and cytokines,will be used to treat the transfected cells. The level of Luciferaseactivity is then determined using commercially available Luciferaseassay systems (Promega) according to manufacturer's instruction toidentify agents that either increase or decrease the expression ofscurfin.

In an alternative approach, agents such as those described above areincubated with primary T cells under conditions that allow for themodulation of scurfin expression. The scurfin expression will bemeasured using the RT-PCR method described above in Example 21. Agentsidentified by either of the above methods will be used directly for thetreatment of an autoimmune disease. Alternatively, T cells will beisolated from patients of an autoimmune disease, treated with thespecific agents identified above to induce scurfin expression andtransferred back into the patients to suppress the activation of other Tcells.

In summary, the results of the Examples show that Foxp3 expression ispredominantly seen in CD4+CD25+ T-reg subset and correlates with a basallevel of regulatory activity. Over-expression of this gene can confer aregulatory function on CD4+ cells that lack CD25, indicating that thisfactor may be directly involved in commitment to this functionallineage.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A method for identifying a compound that modulates the level ofexpression of scurfin comprising the steps of: (a) providing acomposition comprising a reporter gene ligated to a scurfin promoter;(b) contacting the composition with a test compound; (c) determining thelevel of reporter gene expression; and (d) comparing the level ofreporter gene expression in (c) with the predetermined level ofexpression and thereby determining if the test compound modulates theexpression of scurfin.
 2. The method of claim 1, wherein the level ofscurfin expression is decreased.
 3. The method of claim 1, wherein thelevel of scurfin expression is increased.
 4. The method of claim 1,wherein the test compound is selected from the group consisting of amonoclonal antibody, a polyclonal antibody, a peptide, a small molecule,an organic molecule, a natural product, a peptide, an oliciosaccharide,a nucleic acid, a lipid, and an antibody or binding fragment thereof. 5.(canceled)
 6. The method of claim 1, wherein the test compound is from alibrary of compounds.
 7. The method of claim 6, wherein the library isselected from the group consisting of a random peptide library, acombinatorial library, an oligosaccharide library and a phage displaylibrary.
 8. A compound identified according to the method of claim
 1. 9.A method for suppressing an immune response in a mammal comprisingcontacting T cells of the mammal with a compound that increases scurfinexpression in the T cell, wherein an immune response is suppressed. 10.A method for enhancing an immune response in a mammal comprisingcontacting T cells of the mammal with a compound that decreases scurfinexpression in the T cell, wherein an immune response is enhanced.
 11. Amethod of claim 9 wherein said immune response is an autoimmuneresponse, and wherein said contacting results from administering saidcompound to said mammal which increases scurfin expression in said Tcells, thereby inhibiting an autoimmune response by the subject andwherein the autoimmune response is selected from the group consisting ofInflammatory Bowel Disease, Multiple Sclerosis, Rheumatoid Arthritis,Psoriasis, Diabetes, and Asthma.
 12. (canceled)
 13. A method of claim 10wherein said contacting results from administering to said mammal saidcompound which decreases scurfin expression, thereby enhancing an immuneresponse to a disease in the subject.
 14. The method of claim 13,wherein the disease is selected from the group consisting of AIDS andcancer.
 15. A method of claim 9 wherein said immune response contributesto graft versus host disease wherein said contacting comprisesadministering to the mammal said compound that increases scurfinexpression in said T cells, thereby inhibiting graft versus host diseasein the subject.
 16. A method of claim 9 wherein said method comprises:(a) isolating T cells from said mammal; (b) transducing the T cells withthe scurfin gene; (c) expanding the transduced T cells; and (d)reintroducing the transduced T cells into said mammal, wherein anautoimmune response in the patient is inhibited.
 17. The method of claim16 wherein the T cells are CD4+CD25+ regulatory T cells.
 18. The methodof claim 16, wherein the autoimmune disease is selected from the groupconsisting of Inflammatory Bowel Disease, Multiple Sclerosis, RheumatoidArthritis, Psoriasis, Diabetes, and Asthma.
 19. The method of claim 16,wherein the transduction vector is a retroviral vector.
 20. A method ofclaim 10 wherein said method comprises: (a) isolating T cells from saidmammal; (b) transfecting the T cells with a compound that inhibitsscurfin expression; (c) expanding the transfected T cells; and (d)reintroducing the transfected T cells into said mammal, wherein animmune response to a disease in the patient is enhanced.
 21. The methodof claim 20 wherein the compound is an anti-sense molecule.
 22. Themethod of claim 20, wherein the disease is selected from the groupconsisting of AIDS and cancer.